Methods for Examining Podocyte Foot Processes in Human Renal Samples Using Conventional Optical Microscopy

ABSTRACT

The invention provides a method for preparing an expanded renal (kidney) tissue sample suitable for microscopic analysis. Expanding the kidney sample can be achieved by binding, e.g., anchoring, key biomolecules to a polymer network and swelling, or expanding, the polymer network, thereby moving the biomolecules apart as further described herein. As the biomolecules are anchored to the polymer network, isotropic expansion of the polymer network retains the spatial orientation of the biomolecules resulting in an expanded, or enlarged, kidney sample.

RELATED APPLICATION

This application is a continuation of International Application No.PCT/US18/19694, which designated the United States and was filed on Feb.26, 2018, published in English, which claims the benefit of U.S.Provisional Application No. 62/463,251, filed on Feb. 24, 2017. Theentire teachings of the above applications are incorporated herein byreference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant Nos.NS087724 and TR001102 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

In pathology, examination of cellular structures and molecularcomposition using diffraction-limited microscopy has long been key tothe diagnosis of a wide variety of pre-disease and disease states.

Kidney podocytes and their foot processes (FP) are a key component ofthe ultrafiltration system in the glomerulus where they comprise thefiltration barrier together with endothelial cells and the glomerularbasement membrane (GBM). Podocytes have unique anatomy as characterizedby a cell body, major (primary and secondary) FPs, and branches of minor(tertiary) FPs that look like interdigitating fingers of two, attachedto the GBM. This unique ultrafine structure functions as primaryfiltrate to allow water, solutes, and small proteins to pass through thecapillary lumen into Bowman's space. It has been increasingly acceptedthat podocyte function and structure are key factors of integrity ofglomerular filtration barrier. Proteinuric kidney diseases are typicallyassociated with various degrees of podocyte membrane remodeling (FPeffacement and/or SD disruption) driven by a rearrangement of thepodocyte microfilament system, such as FSGS and minimal change disease(Wiggins, R. C. The spectrum of podocytopathies: A unifying view ofglomerular diseases. Kidney International 71, 1205-1214 (2007)).High-grade, nephrotic-range proteinuria, such as Glomerular diseases,are preceded by ultrastructural changes in the FP morphology. In thecase of minimal change disease, that may be the only detectable anatomicabnormality. Due to the small size of tertiary FPs, these lesions cannotbe observed by conventional optical microscopy. Our capability to assesstheir pathological alteration relies on electron microscopy (EM).However, Ems are not common imaging equipment in hospitals and evenresearch labs. They are difficult to practice, slow and expensive. Otherimaging technologies, such as super-resolution optical microscopies, arealso very complex and expensive, therefore not readily accessible inclinical settings. Thus, there is a need for higher resolutionmicroscopy that can work with current diffraction limited microscopesand can optically magnify such renal samples with nanoscale precision.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale emphasis instead being placed upon illustrating theprinciples of the invention.

FIG. 1A through FIG. 1M depict the design and validation of expansionpathology (ExPath). (A) Schematic of ExPath workflow. (B) Pre-expansionimage of a 1.5 mm core of normal human breast tissue acquired with awidefield epifluorescent microscope, and stained with DAPI as well asmultiple antibodies. Blue, DAPI; green, vimentin; red, voltage-dependentanion channel (VDAC). (C) ExPath image of the sample of B, on the samescope. (D and E) Root mean square (RMS) length measurement error as afunction of measurement length for pre- vs post-expansion images (bluesolid line, mean of DAPI channel; green solid line, mean of vimentinchannel; shaded area, standard error of mean; n=3 different patients.Average expansion factor: 4.3 (SD: 0.3)). (F) Super-resolutionstructured illumination microscopy (SR-SIM) image of normal human breasttissue, stained with DAPI and multiple antibodies. Blue, DAPI; green,vimentin; red, keratin-19 (KRT19). (G) ExPath image of the sample of Facquired with a spinning disk confocal microscope. (H and I) RMS lengthmeasurement error as a function of measurement length for ExPath vs SIMimages of human breast tissue (blue solid line, mean of DAPI channel;red solid line, mean of KRT19 channel; shaded area, standard error ofmean; n=5 fields of view from samples from 4 different patients. Averageexpansion factor: 4.0 (SD:0.2)). (J) Hematoxylin and eosin (H&E) stainedhuman breast sample with atypical ductal hyperplasia (ADH). Inset (upperleft) is a magnified view of the area framed by the dotted line. (K)ExPath widefield fluorescent image of the sample of J, stained withantibodies against Hsp60 (red) vimentin (green), and DAPI (blue). (L)ExPath widefield fluorescent image of a human breast sample without HER2amplification. Blue, anti-HER2 (not visible); gray, DAPI; green, DNAFISH against chromosome 17 centrosome; red, DNA FISH against HER2. (M)ExPath widefield fluorescent image of a human breast cancer sample withHER2 amplification, stained as in L. Scale bars: (B) 200 μm, (C) 220 μm(physical size post-expansion, 900 μm; expansion factor 4.1), (F) 10 μm,(G) 10 μm (physical size post-expansion, 43 μm, expansion factor 4.3).(J) 5 μm; inset 1 μm (K) 5 μm; inset 1 μm (physical size post-expansion,23 μm; inset, 4.6 μm; expansion factor 4.6). (L) and (M), physical sizepost-expansion 20 μm.

FIG. 2 depicts ExPath imaging of a wide range of human tissue samples.Images of various tissue types for both normal (left images) andcancerous (right images) tissues from human patients. From top tobottom, different rows show different tissue types as labeled (e.g.,prostate, lung, breast, etc.). Within each block of images for a giventissue x disease type, there are 5 images shown. The leftmost of the 5images shows a core from a tissue microarray (scale bar, 200 μm). Themiddle column within the 5 images shows two images, the top of which isa small field of view (scale bar, 10 μm), and the bottom of which zoomsinto the area flagged in the top image by a white box (scale bar, 2.5μm). The right column within the 5 images shows the same fields of viewas in the middle column, but post-expansion (yellow scale bar, top10-12.5 bottom 2.5-3.1 μm; physical size post-expansion, top 50 μm,bottom 12.5 μm; expansion factors 4.0-5.0×, see Table 2 for the detail).The samples were stained with DAPI and multiple antibodies. Blue, DAPI;green, vimentin; red, KRT19.

FIG. 3AI to FIG. 3L depicts conditions that affect the successfulexpansion of human tissues. (A) Images of human skin samples stainedwith DAPI (grey) and antibodies against vimentin (green) and ACTA4(red). The samples were digested with 8 units/mL proteinase K solutioncontaining 25 mM Tris (pH 8), 1 mM EDTA, 0.25% Triton X-100, and 0.4 MNaCl at 60° C. for 0.5 hour (i-iii) or 2 hours (iv-vi). (i and iv)Photograph of human skin-hydrogel hybrid sample in PBS buffer, afterdigestion for 0.5 hour (i) or 2 hours (iv); (ii) Pre-expansionwide-field fluorescent image from the sample of (i). (iii)Post-expansion wide-field fluorescent image from the sample of (i), witha dashed orange box highlighting regions with autofluorescence in theDAPI channel and distorted vimentin networks post-expansion. (v) Same as(ii) for the sample in (iv). (vi) Same as (iii) for the sample of (iv)with a dashed orange box highlighting regions with autofluorescence inthe DAPI channel. (B) Similar to (A), except that the samples weredigested with 8 units/mL proteinase K solution containing 25 mM Tris (pH8), 25 mM EDTA, 0.25% Triton X-100, and 0.4 M NaCl, at 60° C. for 0.5hour (i-iii) and 2 hours (iv-vi). (C) Photographs of a human liversample digested with a 1 mM EDTA-based protocol (8 units/mL proteinase Ksolution containing 25 mM Tris (pH 8), 1 mM EDTA, 0.25% Triton X-100,and 0.4 M NaCl at 60° C. for 0.5 hours and 2 hours. (D) Photographs of ahuman liver sample digested with a 25 mM EDTA-based protocol (8 units/mLproteinase K solution containing 25 mM Tris (pH 8), 25 mM EDTA, 0.25%Triton X-100, and 0.4 M NaCl at 60° C. for 0.5 hours and 2 hours. (E)Pre-expansion wide-field fluorescent image of human liver sample stainedwith DAPI (grey) and antibody against ACTA4 (red). The same was digestedwith a 1 mM EDTA-based protocol for 1 hour. (F) Post-expansionwide-field fluorescent image of the same sample as in E. White dashedline outlines an out-of-focus region caused by distortion. (G)Pre-expansion wide-field fluorescent image of human liver sample stainedwith DAPI (grey) and antibody against ACTA4 (red). The sample wasdigested with a 25 mM EDTA-based protocol for 0.5 hour. (H)Post-expansion wide-field fluorescent image of the same sample as in G.(I) Post-expansion confocal image of human lymph node tissue withinvaded breast cancer stained with DAPI (blue) and antibody againstvimentin (green), and treated with a 1 mM EDTA-based protocol for 3hours. (J) Post-expansion confocal image of the same tissue as in I,treated with a 25 mM EDTA-based protocol. (K) Post-expansion confocalimage of normal human kidney tissue fixed with acetone, and stained withantibody against collagen IV, and treated with 0.1 mg/ml Acryloyl-Xprior to in situ polymerization. Cracks are indicated by white arrows.(L) Post-expansion confocal image of the same sample as in K, treatedwith 0.03 mg/ml Acryloyl-X prior to in situ polymerization. Scale bars:Aii and iii, 9.2 μm (physical size: 40 μm, expansion factor: 4.33). Avand vi, 9.4 μm (physical size: 40 μm, expansion factor: 4.28). Bii andiii, 9 μm (physical size: 40 μm, expansion factor: 4.41). By and vi, 8.9μm (physical size: 40 μm, expansion factor: 4.51). E and F, 119 μm(physical size: 500 μm, expansion factor: 4.22); G and H, 109 μm(physical size: 500 μm, expansion factor: 4.58). (I-L) 40 μm, physicalsize.

FIG. 4 Representative images of a benign breast lesion from aHematoxylin and Eosin (H&E) stained slide before and after treatment ofexpansion. For the post-expansion images, blue: DAPI, green: Vimentin,red: Hsp60 (mitochondria). Scale bar: top left, 15 μm; top right, 65 μm;bottom left, 2.5 μm; bottom right, 12 μm.

FIG. 5A through FIG. 5G ExPath analysis of clinically relevant nanoscalechanges: kidney podocyte foot process effacement. (A) Pre-expansionconfocal image of a normal human kidney sample showing part of aglomerulus acquired with a spinning disk confocal microscope, andstained with DAPI as well as multiple antibodies. Blue, vimentin; green,actinin-4; red, collagen IV; grey, DAPI. Orange dotted line indicateswhere a line cut is analyzed in C. (B) ExPath image of the same samplewith the same microscope. Red dotted line indicates where a line cut isanalyzed in C. (C) Profiles of actinin-4 intensity along the orange andred dotted lines of (A) and (B). (D) Electron micrograph of a clinicalbiopsy sample from a normal human kidney. Inset, zoom-in to regionoutlined by black box (dotted lines); dotted line within the insetindicates where a line cut is analyzed below. Below, electron micrographfeature intensity along the line cut of the inset. (E) ExPath image of aclinical kidney biopsy sample from the same patient analyzed in (D), andstained as in (A). Blue, vimentin; green, actinin-4; red, collagen IV;grey, DAPI. Inset, zoom-in to region outlined by white box (dottedlines); dotted line within the inset indicates where a line cut isanalyzed below. Below, actinin-4 intensity along the line cut of theinset. (F) As in D, but for a patient with MCD. (G) As in E, but for thesame patient as in F. Scale bars: (A) 5 μm, (B) 5 μm (physical sizepost-expansion, 23.5 μm; expansion factor: 4.7), (D) 1 μm; inset, 200nm, (E) 1 μm (physical size post-expansion, 4.3 μm; expansion factor:4.3); inset, 200 nm, (F) 1 μm, inset, 200 nm, (G) 1 μm (Physical sizepost-expansion, 4.2 μm; expansion factor: 4.2); inset, 200 nm.

FIG. 6A and FIG. 6B demonstrates that Heat treatment improvesimmunostaining of Actinin-4 on human kidney samples. (A) widefieldfluorescent images of human kidney sections with and without heattreatment in citrate buffer. (B) zoom-in regions corresponding to theregions indicated by the dashed yellow rectangles in the left panels.Scale bars: 1 mm (A), 200 μm (B).

FIG. 7A through FIG. 7H demonstrates that anti-Actinin-4 specificallystains tertiary podocyte foot processes. (A-D) Post-expansion widefieldimages of a human kidney section stained with DAPI and antibodies. Blue,vimentin; Green, actinin-4; Red, synaptopodin; Grey, DAPI. (E) Mergedimage of (A-D). (F and G) Magnified regions in actinin-4 (F) andsynaptopodin (G) channels from the same sample as taken from the whitedashed squares in B and C. (H) Profiles of fluorescent intensities takenalong the white dashed line cuts of F and G. Green, actinin-4 red,synaptopodin. Scale bars: 1 μm (4.5 μm physical size, expansion factor4.5).

FIG. 8A through FIG. 8H Immunostaining images of kidney FFPE samples.(A). Post-expansion widefield image of a normal human kidney FFPE sampletreated with a citrate antigen retrieval method (20 mM sodium citrate,pH 8.0), with magnified region (boxed line) zoomed in in (B). (C)Post-expansion widefield image of a normal human kidney FFPE sampletreated with a Tris-EDTA antigen retrieval method (10 mM Tris base, 1 mMEDTA solution, 0.05% Tween 20, pH 9.0), with magnified region (boxedline) zoomed in in (D). (E) Post-expansion confocal image of a normalhuman kidney FFPE sample treated with a citrate antigen retrievalmethod, with magnified region (boxed line) zoomed in in (F). (G)Post-expansion confocal image of a human kidney minimal change diseaseFFPE sample treated with a citrate antigen retrieval method, withmagnified region (boxed line) zoomed in in (H). All the samples werestained with DAPI (gray) and antibodies against vimentin (blue),actinin-4 (green) and collagen IV (red). Scale bars: (A), (C), (E) and(G), 40 μm. (B), (D), (F), and (H), 8 μm.

FIG. 9A through FIG. 9C ExPath significantly improves computationaldiagnosis in early breast lesions. (A) Automated segmentation framework:steps of the image pre-processing and nuclei segmentation pipeline:noise removing using rolling ball correction→enhancing contrast byhistogram equalization→nuclei segmentation by minimum errorthresholding→seed detection by multi-scale Laplacian of Gaussian (LoG)filter→nuclei splitting by marker-controlled watershed. (B)Computational detection and segmentation of the nuclei is significantlymore accurate in expanded samples as compared to pre-expanded samples:example of atypical ductal hyperplasia (ADH); green, true positive; red,false negative; blue, false positive). (C) Classification models werebuilt using L1-regularized logistic regression (GLMNET classifier).Classification accuracy was measured as the area under the receiveroperator curve (AUC) achieved by the classification model incross-validation. ExPath improves automated diagnosis in early breastneoplasia lesions: we applied this image classification framework onboth pre-expanded H&E and expanded images for computationaldifferentiation of normal, benign and pre-invasive malignant breastdiseases. Both data sets consist of 105 images, containing 36 normalbreast tissue images, 31 benign breast tissue images (15 UDH and 16 ADH)and 38 non-invasive breast cancer tissue images (DCIS). Averageexpansion factor: 4.8 (SD: 0.3). *P<0.05, bootstrapped paired t-test. Pvalue for each binary comparison: Normal vs. UDH (0.17); Normal vs. ADH(0.34); Normal vs. DCIS (0.24); UDH vs. ADH (0.02); UDH vs. DCIS (0.01);ADH vs. DCIS (0.24).

DETAILED DESCRIPTION

Method and compositions are provided for imaging cell and tissue samplesby physically, rather than optically, magnifying them. Internationalpatent application serial number PCT/US15/16788, filed on Feb. 20, 2015,which is incorporated herein by reference, teaches that the resolutionof conventional optical microscopy can be increased 4-5 fold byphysically expanding specimens, a processed termed ‘expansionmicroscopy’ (ExM). Briefly, biological specimens are embedded in aswellable hydrogel material, subjected to a treatment to disrupt nativebiological networks, and then expanded. The advantages of ExM includetissue clearing, resolution improvement, and higher tolerance tosectioning error due to the specimen expansion in the z-axis. However,the ExM process was limited in the increased fold of physical expansionand in that the degree of expansion of one sample to the next wasinconsistent.

The invention provides expansion pathology method (ExPath), a simple andversatile method for optical interrogation of clinical biopsy sampleswith nanoscale precision and molecular identity. ExPath is capable ofprocessing the majority of clinical samples currently used in pathologyincluding formalin-fixed paraffin-embedded (FFPE), hematoxylin and eosin(H&E)-stained, and/or fresh frozen tissue specimens and thus enablesnanoscale imaging without the need for hardware beyond that found inconventional laboratories. ExPath functions well on a wide diversity oftissue types and has immediate clinical application in the diagnosis ofdiseases known to exhibit nanoscale pathology.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” are defined to mean “one or more” and include the pluralunless the context clearly dictates otherwise.

It is further noted that the claims can be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which can be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentteachings. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Podocytes (or visceral epithelial cells) are cells of the visceralepithelium in the kidneys and form a crucial component of the glomerularfiltration barrier, contributing size selectivity and maintaining amassive filtration surface. Podocyte loss can lead to proteinuria, andin some disease states to glomerulosclerosis.

The invention provides a method for preparing an expanded renal (kidney)tissue sample. The expanded kidney sample is suitable for microscopicanalysis. By “microscopic analysis” it is meant the analysis of a sampleusing any technique that provides for the visualization of aspects of asample that cannot be seen with the unaided eye, i.e., that are notwithin the resolution range of the normal eye. By “preparing an expandedkidney sample” it is generally meant that the kidney sample isphysically expanded, or enlarged, relative to the sample prior to beexposed to the method(s) described herein. Expanding the kidney samplecan be achieved by binding, e.g., anchoring, key biomolecules to apolymer network and swelling, or expanding, the polymer network, therebymoving the biomolecules apart as further described below. As thebiomolecules are anchored to the polymer network isotropic expansion ofthe polymer network retains the spatial orientation of the biomoleculesresulting in an expanded, or enlarged, kidney sample.

In one embodiment, the method for preparing an expandable kidney tissuesample comprises the steps of staining a kidney tissue sample with footprocess-specific antibodies; permeating the sample with precursors of aswellable polymer; polymerizing the precursors to form a swellablepolymer within the sample; and incubating the sample with a non-specificprotease in a buffer comprising a metal ion chelator, a non-ionicsurfactant, and a monovalent salt. The expandable kidney tissue samplecan be expanded by contacting the swellable polymer with a solvent orliquid to cause the swellable polymer to swell. In one embodiment, priorto staining the sample, the sample is subjected to any suitable antigenretrieval process known to one of skill in the art and as furtherdescribed below. In one embodiment, the method further comprises thestep of treating the specimen with a bifunctional crosslinker. In oneembodiment, the method further comprises the step of anchoring theantibodies and/or proteins of the foot processes to the swellablepolymer. In one embodiment, the method comprises incubating the samplewith 1-100 U/ml of a non-specific protease in a buffer having a pHbetween about 4 and about 12, the buffer comprising about 5 mM to about100 mM metal ion chelator, about 0.1% to about 1.0% non-ionicsurfactant, and about 0.05 M to about 1 M monovalent salt. In oneembodiment, the sample is incubated for about 0.5 to about 3 hours atabout 50° C. to about 70° C.

In one embodiment, the invention provides a method for examiningpodocyte foot processes. The method comprises the steps of staining akidney tissue sample with foot process-specific antibodies; permeatingthe sample with precursors of a swellable polymer; polymerizing theprecursors to form a swellable polymer within the sample; incubating thesample with a non-specific protease in a buffer comprising a metal ionchelator, a non-ionic surfactant, and a monovalent salt; and contactingthe swellable polymer with a solvent or liquid to cause the swellablepolymer to swell. By swelling the polymer, the kidney sample isphysically expanded, or enlarged, relative to the sample prior toswelling. The expanded sample can then be subjected to microscopicanalysis. In one embodiment, the method comprises incubating the samplewith 1-100 U/ml of a non-specific protease in a buffer having a pHbetween about 4 and about 12, the buffer comprising about 5 mM to about100 mM metal ion chelator, about 0.1% to about 1.0% non-ionicsurfactant, and about 0.05 M to about 1 M monovalent salt. In oneembodiment, the sample is incubated for about 0.5 to about 3 hours atabout 50° C. to about 70° C.

In one embodiment, prior to the staining the sample, the sample issubjected to any suitable antigen retrieval process known to one ofskill in the art and as further described below. In one embodiment, themethod further comprises the step of treating the specimen with abifunctional crosslinker. In one embodiment, the method furthercomprises the step of anchoring the antibodies and/or proteins of thefoot processes to the swellable polymer.

In one embodiment, the invention provides a method for diagnosing kidneydisease. The method comprises the steps of staining a kidney tissuesample with foot process-specific antibodies; permeating the sample withprecursors of a swellable polymer; polymerizing the precursors to form aswellable polymer within the sample; incubating the sample with anon-specific protease in a buffer comprising a metal ion chelator, anon-ionic surfactant, and a monovalent salt; and contacting theswellable polymer with a solvent or liquid to cause the swellablepolymer to swell. By swelling the polymer, the kidney sample isphysically expanded, or enlarged, relative to the sample prior toswelling. The expanded sample can then be subjected to microscopicanalysis. In one embodiment, the method comprises incubating the samplewith 1-100 U/ml of a non-specific protease in a buffer having a pHbetween about 4 and about 12, the buffer comprising about 5 mM to about100 mM metal ion chelator, about 0.1% to about 1.0% non-ionicsurfactant, and about 0.05 M to about 1 M monovalent salt. In oneembodiment, the sample is incubated for about 0.5 to about 3 hours atabout 50° C. to about 70° C.

In one embodiment, prior to the staining the sample, the sample issubjected to any suitable antigen retrieval process known to one ofskill in the art and as further described below. In one embodiment, themethod further comprises the step of treating the specimen with abifunctional crosslinker. In one embodiment, the method furthercomprises the step of anchoring the antibodies and/or proteins of thefoot processes to the swellable polymer.

In one embodiment, the invention provides a method for analyzing footprocess effacement. The method comprises the steps of staining a kidneytissue sample with foot process-specific antibodies; permeating thesample with precursors of a swellable polymer; polymerizing theprecursors to form a swellable polymer within the sample; incubating thesample with a non-specific protease in a buffer comprising a metal ionchelator, a non-ionic surfactant, and a monovalent salt; and contactingthe swellable polymer with a solvent or liquid to cause the swellablepolymer to swell. By swelling the polymer, the kidney sample isphysically expanded, or enlarged, relative to the sample prior toswelling. The expanded sample can then be subjected to microscopicanalysis. In one embodiment, the method comprises incubating the samplewith 1-100 U/ml of a non-specific protease in a buffer having a pHbetween about 4 and about 12, the buffer comprising about 5 mM to about100 mM metal ion chelator, about 0.1% to about 1.0% non-ionicsurfactant, and about 0.05 M to about 1 M monovalent salt. In oneembodiment, the sample is incubated for about 0.5 to about 3 hours atabout 50° C. to about 70° C.

In one embodiment, prior to the staining the sample, the sample is heattreated. By “heat treated” it is generally meant any suitable antigenretrieval process known to one of skill in the art and as furtherdescribed below. In one embodiment, the method further comprises thestep of treating the specimen with a bifunctional crosslinker. In oneembodiment, the method further comprises the step of anchoring theantibodies and/or proteins of the foot processes to the swellablepolymer.

The methods of the invention are suitable for diagnosis of podocytediseases or disorders. Podocyte diseases or disorders include but arenot limited to loss of podocytes (podocytopenia), podocyte mutation, anincrease in foot process width, or a decrease in slit diaphragm length.In one aspect, the podocyte-related disease or disorder can beeffacement or a diminution of podocyte density. In another aspect, thediminution of podocyte density could be due to a decrease in a podocytenumber, for example, due to apoptosis, detachment, lack ofproliferation, DNA damage or hypertrophy.

The methods of the invention are suitable for diagnosis by observing andmeasuring the characteristics of visualized tertiary foot processesincluding, but not limited to, general morphology, widths of individualtertiary foot processes, and intervals between two adjacent tertiaryfoot processes.

The terms “biological specimen” or “biological sample” is used herein ina broad sense and is intended to include sources that contain nucleicacids and can be fixed. Exemplary biological samples include, but arenot limited to tissues, including but not limited to, brain, lung,breast, ovary, prostate, testis, stomach, intestine, colon, liver,pancreas, spleen, kidney, heart, muscle, skin, thymus, tonsil tissue.Other exemplary biological samples include, but are not limited to,biopsies, bone marrow samples, organ samples, skin fragments andorganisms. Materials obtained from clinical or forensic settings arealso within the intended meaning of the term biological sample. In oneembodiment, the sample is derived from a human, animal or plant. In oneembodiment, the biological sample is a tissue sample, preferably anorgan tissue sample. In one embodiment, samples are human. The samplecan be obtained, for example, from autopsy, biopsy or from surgery. Itcan be a solid tissue such as, for example, parenchyme, connective orfatty tissue, heart or skeletal muscle, smooth muscle, skin, brain,nerve, kidney, liver, spleen, breast, carcinoma (e.g. bowel,nasopharynx, breast, lung, stomach etc.), cartilage, lymphoma,meningioma, placenta, prostate, thymus, tonsil, umbilical cord oruterus. The tissue can be a tumor (benign or malignant), cancerous orprecancerous tissue. The sample can be obtained from an animal or humansubject affected by disease or other pathology or suspected of same(normal or diseased), or considered normal or healthy. As used herein,the term “fixed biological sample, explicitly excludes cell-freesamples, for example cell extracts, wherein cytoplasmic and/or nuclearcomponents from cells are isolated.

Tissue specimens suitable for use with the methods and systems describedherein generally include any type of tissue specimens collected fromliving or dead subjects, such as, e.g., biopsy specimens and autopsyspecimens. Tissue specimens may be collected and processed using themethods and systems described herein and subjected to microscopicanalysis immediately following processing or may be preserved andsubjected to microscopic analysis at a future time, e.g., after storagefor an extended period of time. In some embodiments, the methodsdescribed herein may be used to preserve tissue specimens in a stable,accessible and fully intact form for future analysis. For example,tissue specimens, such as, e.g., human brain tissue specimens, may beprocessed as described above and cleared to remove a plurality ofcellular components, such as, e.g., lipids, and then stored for futureanalysis.

Tissues that have been preserved, or fixed, contain a variety ofchemical modifications that can reduce the detectability of proteins inbiomedical procedures. In some embodiments, the methods and systemsdescribed herein may be used to analyze a previously-preserved or storedtissue specimen. Previously preserved tissue specimens include, forexample, clinical samples used in pathology including formalin-fixedparaffin-embedded (FFPE), hematoxylin and eosin (H&E)-stained, and/orfresh frozen tissue specimens. If the previously preserved sample has acoverslip, the coverslip should be removed. The sample is treated toremove the mounting medium. Such methods for removing the mountingmedium are well known in the art. For example, treating the sample withxylene to remove paraffin or another hydrophobic mounting medium.Alternatively, if the sample is mounted in a water-based mountingmedium, the sample is treated with water. The sample is then thenrehydrated and subjected to antigen-retrieval. The term “antigenretrieval” refers to any technique in which the masking of an epitope isreversed, and epitope-antibody binding is restored such as, but notlimited to, enzyme induced epitope retrieval, heat induced epitoperetrieval (HIER), or proteolytic induced epitope retrieval (PIER). Forexample, the antigen retrieval treatment can be performed in a 10 mMsodium citrate buffer as well as the commercially available TargetRetrieval Solution (DakoCytomation) or such.

By “biomolecules” it is generally meant, but not limited to, proteins,lipids, steroids, nucleic acids, and sub-cellular structures within atissue or cell.

By “macromolecules” is meant proteins, nucleic acids, or small moleculesthat target biomolecules within the specimen. These macromolecules areused to detect biomolecules within the specimen and/or anchor thebiolmolecules to the swellable polymer. For example, macromolecules maybe provided that promote the visualization of particular cellularbiomolecules, e.g., proteins, lipids, steroids, nucleic acids, etc. andsub-cellular structures. In some embodiments, the macromolecules arediagnostic. In some embodiments, the macromolecules are prognostic. Insome embodiments, the macromolecules are predictive of responsiveness toa therapy. In some embodiments, the macromolecules are candidate agentsin a screen, e.g., a screen for agents that will aid in the diagnosisand/or prognosis of disease, in the treatment of a disease, and thelike.

As an example, the specimen may be contacted with one or morepolypeptide macromolecules, e.g., antibodies, labeled peptides, and thelike, that are specific for and will bind to particular cellularbiomolecules for either direct or indirect labeling by color orimmunofluorescence. By immunofluorescence it is meant a technique thatuses the highly specific binding of an antibody to its antigen orbinding partner in order to label specific proteins or other moleculeswithin the cell. A sample is treated with a primary antibody specificfor the biomolecule of interest. A fluorophore can be directlyconjugated to the primary antibody or peptide. Alternatively, asecondary antibody, conjugated to a detection moiety or fluorophore,which binds specifically to the first antibody can be used. Peptidesthat are specific for a target cellular biomolecule and that areconjugated to a fluorophor or other detection moiety may also beemployed.

In one embodiment, the foot process-specific antibodies are primaryantibodies that are specific for proteins that are abundant in tertiaryfoot processes. In one embodiment, the foot process-specific antibodiesinclude, but are not limited to, alpha-actinin-4, synaptopodin, B7-1,CD2 associated protein (CD2AP), cluster of differentiation 10 (CD10),cortactin, dystroglycan (DG), F-Actin (FAT), Glomerular epithelialprotein 1 (GLEPP1), Microtubule-associated protein 1 light chain 3(MAP-LC3), Myocilin, nephrin-like protein 1 (NEPH1), nephrin,P-cadherin, podocalyxin-like protein in humans (PHM-5), podocin,podoplanin, podocalyxin (PC), T-/H-cadherin (CDH13), 13A antigen,desmin, ezrin, Lmxlb, myocilin, vimentin, zonula occludens-1 (ZO-1), andWilms' tumor-1 protein (WT-1). Antibodies useful in the inventionspecifically bind to any one of these proteins. In one embodiment, twoor more antibodies that bind to the same protein or any combination ofthese proteins can be used in the invention. For example, if twoantibodies are used, the first antibody can specifically bindalpha-actinin-4 and the second antibody can bind any one ofsynaptopodin, B7-1, CD2 associated protein (CD2AP), cluster ofdifferentiation 10 (CD10), cortactin, dystroglycan (DG), F-actin (FAT),glomerular epithelial protein 1 (GLEPP1), microtubule-associated protein1 light chain 3 (MAP-LC3), myocilin, nephrin-like protein 1 (NEPH1),nephrin, P-cadherin, podocalyxin-like protein in humans (PHM-5),podocin, podoplanin, podocalyxin (PC), T-/H-cadherin (CDH13), 13Aantigen, desmin, ezrin, Lmx1b, myocilin, vimentin, zonula occludens-1(ZO-1), or Wilms' tumor-1 protein (WT-1). One skilled in the art caneasily determine any combination of two or more antibodies that arespecific for these proteins or any proteins that are abundant intertiary foot processes.

Another example of a class of agents that may be provided asmacromolecules is nucleic acids. For example, a specimen may becontacted with an antisense RNA that is complementary to andspecifically hybridizes to a transcript of a gene of interest, e.g., tostudy gene expression in cells of the specimen. As another example, aspecimen may be contacted with a DNA that is complementary to andspecifically hybridizes to genomic material of interest, e.g., to studygenetic mutations, e.g., loss of heterozygosity, gene duplication,chromosomal inversions, and the like. The hybridizing RNA or DNA isconjugated to detection moieties, i.e. agents that may be eitherdirectly or indirectly visualized microscopically. Examples of in situhybridization techniques may be found at, for example, Harris andWilkinson. In situ hybridization: Application to developmental biologyand medicine, Cambridge University Press 1990; and Fluorescence In SituHybridization (FISH) Application Guide. Liehr, T, ed., Springer-Verlag,Berlin Heidelberg 1990.

In one embodiment, the biological sample can be labeled or tagged with adetectable label. Typically, the label or tag will bind chemically(e.g., covalently, hydrogen bonding or ionic bonding) to a biomoleculeof the sample, or a component thereof. The detectable label can beselective for a specific target (e.g., a biomarker or class ofmolecule), as can be accomplished with an antibody or other targetspecific binder. The detectable label may comprise a visible component,as is typical of a dye or fluorescent molecule; however, any signalingmeans used by the label is also contemplated. A fluorescently labeledbiological sample, for example, is a biological sample labeled throughtechniques such as, but not limited to, immunofluorescence,immunohistochemical or immunocytochemical staining to assist inmicroscopic analysis. In one embodiment, the detectable label ischemically attached to the biological sample, or a targeted componentthereof. In one embodiment, the detectable label is an antibody and/orfluorescent dye wherein the antibody and/or fluorescent dye, furthercomprises a physical, biological, or chemical anchor or moiety thatattaches or crosslinks the specimen to the swellable polymer, such as aswellable hydrogel. The labeled sample may furthermore include more thanone label. For example, each label can have a particular ordistinguishable fluorescent property, e.g., distinguishable excitationand emission wavelengths. Further, each label can have a differenttarget specific binder that is selective for a specific anddistinguishable target in, or component of the sample.

In one embodiment, the sample contacted with a bi-functional linkerwherein the bi-functional linker comprises a binding moiety and ananchor, wherein the binding moiety binds to biomolecules in the sample.The anchor may be a physical, biological, or chemical moiety thatattaches or crosslinks the sample to the composition, hydrogel or otherswellable material. This may be accomplished by crosslinking the anchorwith the swellable material, such as during or after the polymerization,i.e., in situ formation of the swellable material. The anchor maycomprise a polymerizable moiety. The anchor may include, but is notlimited to, vinyl or vinyl monomers such as styrene and its derivatives(e.g., divinyl benzene), acrylamide and its derivatives, butadiene,acrylonitrile, vinyl acetate, or acrylates and acrylic acid derivatives.The polymerizable moiety may be, for example, an acrylamide modifiedmoiety that may be covalently fixed within a swellable material.

As used herein a bifunctional crosslinker comprises reactive groups tofunctional groups (e.g., primary amines or sulfhydryls) on biomoleculeswithin the sample. The bifunctional crosslinker may be used tochemically modify the amine group of biomolecules with a swellablepolymer functional group, which enables antibodies and other endogenousbiomolecules within the sample to be directly anchored to, orincorporate into, the swellable polymer. In one embodiment, thebifunctional crosslinker is a hetero-bifunctional crosslinker.Hetero-bifunctional crosslinkers possess different reactive groups ateither end of a spacer arm, i.e., atoms, spacers or linkers separatingthe reactive groups. These reagents not only allow for single-stepconjugation of molecules that have the respective target functionalgroup, but they also allow for sequential (two-steps) conjugations thatminimize undesirable polymerization or self-conjugation. Thebi-functional linker may be a small molecule linker or a nucleic acidadaptor.

As used herein, a “nucleic acid adaptor” is a nucleic acid sequencehaving a binding moiety capable of attaching to a target nucleic acidand an anchor moiety capable of attaching to the swellable material.Attaching the nucleic acid adaptor to a target nucleic acid may beaccomplished by hybridization or by ligation in situ. For example, DNAadaptors may be ligated to the 3′ ends of the RNAs in the sample withRNA ligases, such as T4 RNA ligase, or may be attached via a chemicallinker such as a reactive amine group capable of reacting with targetnucleic acid. Acrylamide modified oligonucleotide primers may becovalently fixed within a swellable material such as a polyacrylate gel.As used herein, the term “acrylamide modified” in reference to anoligonucleotide means that the oligonucleotide has an acrylamide moietyattached to the 5′ end of the molecule.

As used herein, a “small molecule linker” is a small molecule having abinding moiety capable of attaching to a target nucleic acid and ananchor moiety capable of attaching to the swellable material. Attachingthe small molecule linker to the target nucleic acid may be accomplishedby hybridization or by a chemical reactive group capable of covalentlybinding the target nucleic acid. For example, LABEL-IT® Amine (MirusBio)is a small molecule with alkylating group that primarily reacts to theN7 of guanine, thereby allowing covalent binding of RNA and DNA. Thesmall molecule linker may be, for example, acrylamide modified andtherefore may be covalently fixed within a swellable material. As usedherein, the term “acrylamide modified” in reference to a small moleculelinker means that the small molecule linker has an acrylamide moiety.

In one embodiment, the bifunctional crosslinker comprises aprotein-reactive chemical moiety and a gel-reactive chemical moiety(i.e., a swellable polymer-reactive chemical moiety). Theprotein-reactive chemical group includes, but is not limited to,N-hydroxysuccinimide (NHS) ester, thiol, amine, maleimide, imidoester,pyridyldithiol, hydrazide, phthalimide, diazirine, aryl azide,isocyanate, or carboxylic acid, which, for example, can be reacted withamino or carboxylic acid groups on proteins or peptides. In oneembodiment, the protein-reactive groups include, but are not limited to,N-succinimidyl ester, pentafluorophenyl ester, carboxylic acid, orthiol. The gel-reactive groups include, but are not limited to, vinyl orvinyl monomers such as styrene and its derivatives (e.g., divinylbenzene), acrylamide and its derivatives, butadiene, acrylonitrile,vinyl acetate, or acrylates and acrylic acid derivatives.

In one embodiment, the chemical to anchor proteins directly to anyswellable polymer is a succinimidyl ester of 6-((acryloyl)amino)hexanoic acid (acryloyl-X, SE; abbreviated “AcX”; Life Technologies).Treatment with AcX modifies amines on proteins with an acrylamidefunctional group. The acrylamide functional groups allow for proteins tobe anchored to the swellable polymer as it is synthesized in situ.

As used herein, the term “attach” or “attached” refers to both covalentinteractions and noncovalent interactions. In certain embodiments of theinvention, covalent attachment may be used, but generally all that isrequired is that the bi-functional linker remain attached to the targetnucleic acid under conditions for nucleic acid amplification and/orsequencing. Oligonucleotide adaptors may be attached such that a 3′ endis available for enzymatic extension and at least a portion of thesequence is capable of hybridizing to a complementary sequence.Attachment can occur via hybridization to the target nucleic acid, inwhich case the attached oligonucleotide may be in the 3′-5′ orientation.Alternatively, attachment can occur by means other than base-pairinghybridization, such as the covalent attachment set forth above. The term“attach” may be used interchangeably herein with the terms,“anchor(ed)”, affix(ed), link(ed) and immobilize(d).

In one embodiment, the proteins of the sample of interest can bemodified with the protein-reactive group and the gel-reactive group inseparate steps using click chemistry. Click chemistry, also referred toas tagging, is a class of biocompatible reactions intended primarily tojoin substrates of choice with specific biomolecules. In this method,proteins of the sample of interest are treated with a protein-reactivegroup comprising a click group and then treated with a gel-reactivegroup comprising a complementary click group. Complementary groupsinclude, but are not limited to, azide groups and terminal alkynes (seee.g., H. C. Kolb; M. G. Finn; K. B. Sharpless (2001). “Click Chemistry:Diverse Chemical Function from a Few Good Reactions”. Angewandie ChemieInternational Edition. 40(11): 2004-2021, which is incorporated hereinby reference).

The biological specimen is embedded in a swellable polymer. By“swellable polymer” it is meant hydrophilic monomers, prepolymers, orpolymers that can be crosslinked, or “polymerized”, to form athree-dimensional (3D) polymer network, which expands when contactedwith a liquid, such as water or other solvent. For example, one or morepolymerizable materials, monomers or oligomers can be used, such asmonomers selected from the group consisting of water soluble groupscontaining a polymerizable ethylenically unsaturated group. Monomers oroligomers can comprise one or more substituted or unsubstitutedmethacrylates, acrylates, acrylamides, methacrylamides, vinylalcohols,vinylamines, allylamines, allylalcohols, including divinyliccrosslinkers thereof (e.g., N,Nalkylene bisacrylamides). Precursors canalso comprise polymerization initiators and crosslinkers.

In one embodiment, the swellable material uniformly expands in 3dimensions. Additionally, or alternatively, the material is transparentsuch that, upon expansion, light can pass through the sample. In oneembodiment, the swellable polymer is a swellable hydrogel. In oneembodiment, the swellable material is formed in situ from precursorsthereof.

By “precursors of a swellable polymer”, “hydrogel subunits” or “hydrogelprecursors” is meant hydrophilic monomers, prepolymers, or polymers thatcan be crosslinked, or “polymerized”, to form a three-dimensional (3D)hydrogel network. In one embodiment the swellable polymer is apolyelectrolyte. In one embodiment, the swellable polymer ispolyacrylate or polyacrylamide and copolymers or crosslinked copolymersthereof.

Without being bound by scientific theory, it is believed that thisfixation of the biological specimen in the presence of hydrogel subunitscrosslinks the biomolecules of the specimen to the hydrogel subunits,thereby securing molecular components in place, preserving the tissuearchitecture and cell morphology.

The precursors of the swellable polymer may be delivered to thebiological specimen by any convenient method including, but not limitedto, permeating, perfusing, infusing, soaking, adding or otherintermixing the sample with the precursors of swellable material. Inthis manner, the biological specimen is saturated with precursors of theswellable material, which flow between and around biomoleculesthroughout the specimen.

Following permeating the specimen, the swellable polymer precursors arepolymerized, i.e., covalently or physically crosslinked, to form apolymer network. The polymer network is formed within and throughout thespecimen. In this manner, the biological specimen is saturated with theswellable material, which flow between and around biomoleculesthroughout the specimen.

Polymerization may be by any method including, but not limited to,thermal crosslinking, chemical crosslinking, physical crosslinking,ionic crosslinking, photo-crosslinking, irradiative crosslinking (e.g.,x-ray, electron beam), and the like, and may be selected based on thetype of hydrogel used and knowledge in the art. In one embodiment, thepolymer is a hydrogel. Once polymerized, a polymer-embedded biologicalspecimen is formed.

In some embodiments, native proteins anchored to the swellable polymerperfused throughout the sample as described herein can retain epitopefunctionality and be labeled post-expansion if the nonspecificproteolysis of ExM is replaced with modified post-gelationhomogenization treatments. Such approaches may overcome the limitationsinherent to delivering antibodies in the crowded environment of nativetissue.

By embedding a specimen in a swellable polymer that physically supportsthe ultrastructure of the specimen this technology preserves thebiomolecules (e.g., proteins, small peptides, small molecules, andnucleic acids in the specimen) in their three-dimensional distribution,secured by the polymer network. By bypassing destructive sectioning ofthe specimen, subcellular structures may be analyzed. In addition, thespecimen can be iteratively stained, unstained, and restained with otherreagents for comprehensive analysis.

After the biological sample has been anchored to the swellable polymer,the specimen is subjected to a disruption of the endogenous biologicalmolecules (or the physical structure of the biological sample), leavingthe macromolecules, e.g., label or tag, that preserve the information ofthe targeted biological molecules intact and anchored to the swellablepolymer. In this way, the mechanical properties of thespecimen-swellable polymer complex are rendered more spatially uniform,allowing greater and more consistent isotropic expansion.

The disruption of the endogenous physical structure of the specimen orof the endogenous biological molecules of the biological specimengenerally refers to the mechanical, physical, chemical, biochemical or,enzymatic digestion, disruption or break up of the sample so that itwill not resist expansion. In one embodiment, a non-specific protease isused to homogenize the sample-swellable polymer complex.

In one embodiment, the non-specific protease is in a buffer having a pHfrom about 4 to about 12. Any suitable buffer agent can be usedincluding, but not limited to, Tris, citrate, phosphate, bicarbonate,MOPS, borate, TAPS, bicine, Tricine, HEPES, TES, and MES. In oneembodiment, the method comprises incubating the sample with anon-specific protease in a buffer comprising a metal ion chelator, anon-ionic surfactant, and a monovalent salt. In one embodiment, thebuffer comprises about 1 U/ml to about 100 U/ml of a non-specificprotease; about 5 mM to about 100 mM metal ion chelator; about 0.1% toabout 1.0% nonionic surfactant; and about 0.05 M to about 1 M monovalentsalt. In one embodiment, the sample is incubated in the buffer for about0.5 to about 3 hours at about 50° C. to about 70° C. In one embodiment,the sample is incubated in the buffer until the sample is completelydigested.

Non-specific proteases are well known to those of skill in the art.Non-specific proteases include, but are not limited to, proteinase K,Subtilisin, pepsin, thermolysin, and Elastase. In one embodiment thebuffer comprises about 1 U/ml to about 100 U/ml of a non-specificprotease. In one embodiment the buffer comprises about 1 U/ml to about50 U/ml of a non-specific protease. In one embodiment the buffercomprises about 1 U/ml to about 25 U/ml of a non-specific protease. Inone embodiment the buffer comprises about 1 U/ml to about 10 U/ml of anon-specific protease.

Chelating agents are well known to those of skill in the art. Chelatingagents include, but are not limited to, EDTA, EGTA, EDDHA, EDDS, BAPTAand DOTA. In one embodiment the buffer comprises about 5 mM to about 100mM of a metal ion chelator. In one embodiment the buffer comprises about5 mM to about 75 mM of a metal ion chelator. In one embodiment thebuffer comprises about 5 mM to about 50 mM of a metal ion chelator.

Nonionic surfactant is well known to those of skill in the art. Nonionicsurfactants include, but are not limited to, Triton X-100, Tween 20,Tween 80, Sorbitan, Polysorbate 20, Polysorbate 80, PEG, Decylglucoside, Decyl polyglucose and cocamide DEA. In one embodiment thebuffer comprises about 0.1% to about 1.0% nonionic surfactant. In oneembodiment the buffer comprises about 0.1% to about 0.75% nonionicsurfactant. In one embodiment the buffer comprises about 0.1% to about0.5% nonionic surfactant. In one embodiment the buffer comprises about0.1% to about 0.3% nonionic surfactant.

Monovalent cation salts are well known to those of skill in the art.Monovalent cation salts contain cations that include, but are notlimited to, Na⁺, K⁺, ammonium, and Cs⁺. In one embodiment, the buffercomprises about 0.05 M to about 1.0 M monovalent salt. In oneembodiment, the buffer comprises about 0.05 M to about 1.0 M monovalentsalt. In one embodiment, the buffer comprises about 0.75 M to about 1.0M monovalent salt. In one embodiment, the buffer comprises about 0.1 Mto about 1.0 M monovalent salt. In one embodiment, the buffer comprisesabout 0.1 M to about 0.7 M monovalent salt. In one embodiment, thebuffer comprises about 0.05 M to about 0.8 M monovalent salt.

It is preferable that the disruption does not impact the structure ofthe swellable polymer but disrupts the structure of the specimen. Thus,the specimen disruption should be substantially inert to the swellablepolymer. The degree of digestion can be sufficient to compromise theintegrity of the mechanical structure of the specimen or it can becomplete to the extent that the specimen-swellable polymer complex isrendered substantially free of the sample.

The specimen-swellable polymer complex is then expanded for example, bycontacting the swellable polymer with a solvent or liquid which is thenabsorbed by the swellable polymer and causes swelling. Where theswellable polymer is water swellable, an aqueous solution can be used.The swelling of the swellable polymer results in the specimen itselfexpanding (e.g., becoming larger). This is because the polymer isembedded throughout the specimen, therefore, as the polymer swells(grows) it expands and causes the anchored biomolecules to pull apart(i.e., move away) from each. In one embodiment, the swellable polymerexpands (swells) isotropically; therefore, the anchored biomoleculesretain the relative spatial orientation within the specimen.

The swollen biological specimen-polymer complex can be imaged on anyoptical microscope, allowing effective imaging of features below theclassical diffraction limit. Since the resultant specimen can betransparent, custom microscopes capable of large volume, wide field ofview, 3D scanning may also be used in conjunction with the expandedsample. The method also provides an optional step comprisingamplification of the detectable label.

Using the described methods, reagents, kits, systems and devices, theordinarily skilled artisan will be able to prepare any biologicalspecimen for microscopic analysis. Methods, reagents, kits, systems anddevices may be used to prepare a specimen from any plant or animal,including but not limited to transgenic animals, e.g., vertebrate orinvertebrate, e.g. insect, worm, xenopus, zebrafish, mammal, e.g.,equine, bovine, ovine, canine, feline, murine, rodent, non-human primateor human. Tissue specimens may be collected from living subjects (e.g.,biopsy samples) or may be collected from dead subjects (e.g., autopsy ornecropsy samples). The specimens may be of any tissue type, e.g.hematopoietic, neural (central or peripheral), glial, mesenchymal,cutaneous, mucosal, stromal, muscle (skeletal, cardiac, or smooth),spleen, reticulo-endothelial, epithelial, endothelial, hepatic, kidney,pancreatic, gastrointestinal, pulmonary, fibroblast, and other celltypes. In some instances, the specimen is the entire organism, e.g. aworm, an insect, a zebrafish. In other instances, the specimen is awhole organ, e.g., the whole brain of a rodent. In other instances, thespecimen is a portion of an organ, i.e. a biopsy, e.g. a biopsy of atransplanted tissue. The specimen may be freshly isolated or preserved,e.g. snap frozen. In some embodiments, the specimen may be a previouslypreserved specimen, such as, e.g., a preserved specimen from a tissuebank, e.g., a preserved specimen of a human brain obtained from a tissuecollection program. In some instances, a specimen may be from a subjectknown to suffer from a specified disease or condition, such as, e.g., asample of brain tissue from an autistic human. In other instances, asample may be from a “normal” subject that does not suffer from aspecific disease or condition. In some instances, a sample may be from atransgenic subject, such as, e.g., a transgenic mouse.

Because the cells and/or biomolecules of the specimen are anchored to aswellable polymer that physically supports the ultrastructure of thespecimen, cellular components (e.g. lipids) that normally providestructural support but that hinder visualization of subcellular proteinsand molecules may be removed while preserving the 3-dimensionalarchitecture of the cells and tissue. This removal renders the interiorof biological specimen substantially permeable to light and/ormacromolecules, allowing the interior of the specimen, e.g. cells andsubcellular structures, to be microscopically visualized withouttime-consuming and disruptive sectioning of the tissue. Additionally,the specimen can be iteratively stained, unstained, and re-stained withother reagents for comprehensive analysis.

The subject methods find many uses. For example, the subject methods maybe applied to preparing specimens for the study of the connectivity ofthe central nervous system. “Connectivity” as used herein generallymeans the connections between neurons, and includes connections at thesingle cell level, e.g., synapses, axon termini, dendritic spines, etc.,as well as connections between groups of neurons and regions of the CNSas major axon tracts, e.g., corpus callosum (CC), anterior commissure(AC), hippocampal commissure (HC), pyramidal decussation, pyramidaltracts, external capsule, internal capsule (IC), cerebral peduncle (CP),etc. A whole brain and/or spinal cord specimen or region thereof (e.g.,cerebrum (i.e., cerebral cortex), cerebellum (i.e., cerebellar cortex),ventral region of the forebrain (e.g., striatum, caudate, putamen,globus pallidus, nucleus accumbens; septal nuclei, subthalamic nucleus);regions and nuclei of the thalamus and hypothalamus; regions and nucleiof the deep cerebellum (e.g., dentate nucleus, globose nucleus,emboliform nucleus, fastigial nucleus) and brainstem (e.g., substantianigra, red nucleus, pons, olivary nuclei, cranial nerve nuclei); andregions of the spine (e.g., anterior horn, lateral horn, posteriorhorn)) may be prepared post-mortem by the subject methods and theconnectivity of the neurons therein microscopically analyzed, e.g.,obtained, stored, rendered, used, and actuated, e.g., to provide thefull connectivity of a brain, e.g., a human brain, after death. Suchstudies will contribute greatly to the understanding of how the braindevelops and functions in health and during disease, and of theunderpinnings of cognition and personality.

As another example, the subject methods may be employed to evaluate,diagnose or monitor a disease. “Diagnosis” as used herein generallyincludes a prediction of a subject's susceptibility to a disease ordisorder, determination as to whether a subject is presently affected bya disease or disorder, prognosis of a subject affected by a disease ordisorder (e.g., identification of cancerous states, stages of cancer,likelihood that a patient will die from the cancer), prediction of asubject's responsiveness to treatment for a disease or disorder (e.g., apositive response, a negative response, no response at all to, e.g.,allogeneic hematopoietic stem cell transplantation, chemotherapy,radiation therapy, antibody therapy, small molecule compound therapy)and use of therametrics (e.g., monitoring a subject's condition toprovide information as to the effect or efficacy of therapy). Forexample, a biopsy may be prepared from a cancerous tissue andmicroscopically analyzed to determine the type of cancer, the extent towhich the cancer has developed, whether the cancer will be responsive totherapeutic intervention, etc.

As another example, a biopsy may be prepared from a diseased tissue,e.g. kidney, pancreas, stomach, etc., to determine the condition of thetissue, the extent to which the disease has developed, the likelihoodthat a treatment will be successful, etc. The terms “treatment”,“treating” and the like are used herein to generally mean obtaining adesired pharmacologic and/or physiologic effect. The effect may beprophylactic in terms of completely or partially preventing a disease orsymptom thereof and/or may be therapeutic in terms of a partial orcomplete cure for a disease and/or adverse effect attributable to thedisease. “Treatment” as used herein covers any treatment of a disease ina mammal and includes: (a) preventing the disease from occurring in asubject which may be predisposed to the disease but has not yet beendiagnosed as having it; (b) inhibiting the disease, i.e., arresting itsdevelopment; or (c) relieving the disease, i.e., causing regression ofthe disease. The therapeutic agent may be administered before, during orafter the onset of disease or injury. The treatment of ongoing disease,where the treatment stabilizes or reduces the undesirable clinicalsymptoms of the patient, is of particular interest. Such treatment isdesirably performed prior to complete loss of function in the affectedtissues. The subject therapy will desirably be administered during thesymptomatic stage of the disease, and in some cases after thesymptomatic stage of the disease. The terms “individual,” “subject,”“host,” and “patient,” are used interchangeably herein and refer to anymammalian subject for whom diagnosis, treatment, or therapy is desired,particularly humans. Examples of diseases that are suitable toevaluation, analysis, diagnosis, prognosis, and/or treatment using thesubject methods and systems include, but are not limited to, cancer,immune system disorders, neuropsychiatric disease,endocrine/reproductive disease, cardiovascular/pulmonary disease,musculoskeletal disease, gastrointestinal disease, and the like.

The subject methods may also be used to evaluate normal tissues, organsand cells, for example to evaluate the relationships between cells andtissues of a normal tissue specimen, e.g., a tissue specimen taken froma subject not known to suffer from a specific disease or condition. Thesubject methods may be used to investigate, e.g., relationships betweencells and tissues during fetal development, such as, e.g., duringdevelopment and maturation of the nervous system, as well as toinvestigate the relationships between cells and tissues afterdevelopment has been completed, e.g., the relationships between cellsand tissues of the nervous systems of a fully developed adult specimen.

The subject methods also provide a useful system for screening candidatetherapeutic agents for their effect on a tissue or a disease. Forexample, a subject, e.g. a mouse, rat, dog, primate, human, etc. may becontacted with a candidate agent, an organ or a biopsy thereof may beprepared by the subject methods, and the prepared specimenmicroscopically analyzed for one or more cellular or tissue parameters.Parameters are quantifiable components of cells or tissues, particularlycomponents that can be accurately measured, desirably in a highthroughput system.

The subject methods may also be used to visualize the distribution ofgenetically encoded markers in whole tissue at subcellular resolution,for example, chromosomal abnormalities (inversions, duplications,translocations, etc.), loss of genetic heterozygosity, the presence ofgene alleles indicative of a predisposition towards disease or goodhealth, likelihood of responsiveness to therapy, ancestry, and the like.Such detection may be used in, for example, diagnosing and monitoringdisease as, e.g., described above, in personalized medicine, and instudying paternity.

In some embodiments, the enlarged sample can be re-embedded in anon-swellable polymer. “Re-embedding” comprises permeating (such as,perfusing, infusing, soaking, adding or other intermixing) the samplewith the non-swellable polymer, preferably by adding precursors thereof.Alternatively or additionally, embedding the sample in a non-swellablepolymer comprises permeating one or more monomers or other precursorsthroughout the sample and polymerizing and/or crosslinking the monomersor precursors to form the non-swellable polymer or polymer. In thismanner the first enlarged sample, for example, is embedded in thenon-swellable polymer. Embedding the expanded sample in a non-swellablepolymer prevents conformational changes during sequencing despite saltconcentration variation. The non-swellable polymer can be charge-neutralhydrogels. For example, it can be polyacrylamide hydrogel, composed ofacrylamide monomers, bisacrylamide crosslinker, ammonium persulfate(APS) initiator and tetramethylethylenediamine (TEMED) accelerator.

In some embodiments, the fixed biological sample is subjected topassivation. As used herein the term “passivation” refers to the processfor rendering the sample less reactive with the components containedwithin the fixative such as by functionalizing the fixative withchemical reagents to neutralize charges within. For example, thecarboxylic groups of acrylates, which may be used in the swellable gel,can inhibit downstream enzymatic reactions. Treating the swellable gelcomposed of acrylate with 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC) and N-Hydroxysuccinimide (NHS) allows primary amines to covalentlybind the carboxylic groups to form charge neutral amides and passivatethe swellable gel.

The innovation enables physical expansion of common clinical tissuespecimen based on the unique physical and chemical properties ofclinical tissue specimens. Clinical tissue specimens are usually highlyfixed, tightly attached on the superfrost glass slides, and embedded inthe paraffin (or stained and mounted in a mounting medium) for long-termstorage. Some clinical tissue specimens are stained with dyes, such ashematoxylin and eosin (H&E), which are incompatible with fluorescenceimaging. To apply ExM to clinical samples, de-paraffinization, antigenretrieval and aggressive protease digestion are integrated in acomprehensive workflow to handle various kinds of common clinicalspecimens. De-paraffinization and antigen retrieval address the recoveryof archived clinical samples, while aggressive protease digestion iscritical for the success of sample expansion, as most of the humantissues contain abundant hard-to-digest structural proteins, such ascollagen and fibronectin, which prevent homogeneous expansion of thesample. Taken together, the present invention allows for the applicationof ExM to the enormous amount of archived clinical samples and enablesuper-resolution optical interrogations of mechanisms of a broad rangeof diseases by conventional optical microscopy.

This invention provides a comprehensive workflow to facilitate expansionof common types of clinical samples for super-resolution molecularimaging. The methods described herein will result in optimal outcomes,such as proper immunostaining, sufficient digestion of tissue, highquality of polymer synthesis, and maintenance of proteins of interestduring expansion.

The invention also describes the reutilization of classic H&E stainedslides for further biomolecular interrogation in nanoscale level. Ingeneral, H&E stained slides are not considered suitable for furtherdownstream processing due to the difficulty in removing the stain andmounting medium. Thus, the invention describes a unique andcost-effective approach to overcome this barrier and enable theextraction of more information from the used H&E slides. In oneembodiment, the method of expanding H&E stained slides for furtherutilization combines xylene-ethanol-water sequential washing, proteinanchoring and in situ polymer synthesis.

Examples Human Samples

The breast pathological specimens used in this study were from thepathology archives of the Beth Israel Deaconess Medical Center and wereused under BIDMC IRB protocol #2013p000410 to AHB. The frozen kidneypathological samples were provided by the Brigham and Woman's archivesunder the BWH IRB protocol #2011P002692 to AW. Other human tissuesamples and tissue microarrays were purchased from commercial sources(see Table 1).

TABLE 1 Human samples purchased from commercial sources SampleManufacturer Catalog No. IHC control tissue array Abcam ab178176 forHER2 molecule Human Adult Normal: Lung (FFPE) US Biomax HuFPT131Multi-tumor tissue array, 95 cases of Abcam ab178234 40 types from 27organs/sites (1.5 mm) Human Adult Normal: Kidney US Biomax HuFTS241(Fresh Frozen slides) Breast hyperplasia tissue US Biomax BR806 array,80 cases/80 cores Breast pre-cancerous disease and cancer US BiomaxBR1003 tissue array (100 cases/101 cores) Breast common disease tissueAbcam ab178113 array of 102 cases (1.5 mm) Breast cancer tissue arraywith progressive Abcam Ab178112 changes, 48 cases, 96 samples (1.5 mm)

The use of unidentified archival specimens do not require informedconsent from the subjects.

Sample Preparation Prior to Antigen Retrieval For Paraffin EmbeddedClinical Samples

In one embodiment the workflow is summarized in FIG. 1. In embodimentswherein the clinical tissue sample is embedded in paraffin,deparaffinization is required. Deparaffinization is performed by placingthe slides in a Coplin jar and sequentially washing the clinical tissuesample using the following solutions: (a) 2×Xylene, (b) 1:1 Xylene:100%Ethanol, (c) 2×100% ethanol, (d) 95% ethanol, (e) 70% ethanol, (f) 50%ethanol, and finally (g) cold tap water. In some embodiments theclinical tissue sample is washed for 3 minutes in each solution. In someembodiments the clinical tissue sample is washed at room temperature. Inembodiments wherein the paraffin embedded clinical tissue sample is on aglass slide, the slide is wash sequentially in the solutions asdescribed herein. In some embodiments, the paraffin-embedded clinicalsamples is part of a tissue microarray. In some embodiments, theparaffin-embedded clinical samples is a tissue microarray. In someembodiments the paraffin-embedded clinical samples are deparaffinizedusing a deparaffinization solution (QIAGEN).

In some embodiments, for formalin-fixed paraffin-embedded (FFPE)clinical samples, samples were placed in a series of solutionssequentially, 3 mins for each step: 2×xylene, 2×100% ethanol, 95%ethanol, 70% ethanol, 50% ethanol, and finally double deionized water.All the steps were performed at room temperature (RT).

For Stained and Mounted Permanent Slides

In embodiments wherein the clinical tissue sample is stained and mountedon a permanent slide the coverslip of the slide is first carefullyremoved. The coverslip can be removed with any appropriate tool, forexample, a razor blade and if the coverslip is difficult to remove,pre-treatment with a xylene solution will help loosen the coverslip. Theslide is then washed sequentially with the xylene-basedde-paraffinization solutions discussed above. In some embodiments, theclinical tissue samples are stained with H&E.

In some embodiments, the slides were treated as FFPE samples discussedabove.

In embodiments where the clinical tissue sample is stained with awater-soluble stain such as eosin, the stain can be washed away withwater. In embodiments where the clinical tissue sample is stained withan insoluble stain, of further stained with an insoluble stain, such ashematoxylin, the insoluble stain can be removed by washing the clinicaltissue sample with the xylene-based de-paraffinization solutionsdiscussed above, or such insoluble stain may remain in the sample, butcan be oxidized and removed after in situ polymer synthesis, digestionand expansion steps.

In some embodiments the insoluble stain can be removed by placing theslide comprising the clinical tissue sample in a 0.1 M HCl solutionuntil the insoluble stain is completely removed. In some embodiments theinsoluble stain is hematoxylin. The drawback of removing the insolublestain in the 0.1 M HCL solution is that the tissue may de-attach fromthe glass slide in the later steps.

In embodiments where the clinical tissue samples are fixed and frozen ona glass slide, the clinical tissue samples are left at room temperatureto allow the freeze cutting medium to melt. If the clinical tissuesample is embedded in paraffin, the sample is de-paraffinized asdiscussed above.

In one embodiment, unfixed frozen tissue slides in optimum cuttingtemperature (OCT) solution (Tissue-Tek) were initially fixed for 5-10min in ice cold acetone at −20° C. before 3× PBS washing. For alreadyfixed and frozen clinical tissue sections, the slides were left at RTfor 2 mins, to let the OCT solution melt and washed 3× with PBSsolution.

Once the clinical tissue samples have been de-paraffinized, or if theclinical tissue samples are not embedded in paraffin but are fixed withparaformaldehyde or similar aldehyde-based chemicals, the clinicaltissue sample then proceeds to the antigen retrieval step.

Antigen Retrieval

In embodiments where the clinical samples that are fixed by formalin orsimilar aldehyde-based chemicals, the clinical tissue samples must betreated with antigen-retrieval procedures prior to the immunostainingstep. If the clinical tissue samples were not paraffin embedded, or havebeen de-paraffinized, and are not fixed by formalin or similaraldehyde-based chemicals, the clinical tissue samples can proceed to theimmunostaining step.

In embodiments where the clinical tissue samples must be treated withantigen retrieval procedures, any heat induced epitope retrieval orenzyme induced epitope retrieval methods known to one skilled in the artor their combination of any kind may be used for antigen retrieval. Forexample, in some embodiments, the clinical tissue samples can be placedin 10 mM sodium citrate solution (pH 8.5), for 5 mins at RT, thentransferred to 10 mM sodium citrate solution (pH 8.5) for 30 mins at80-100° C.

In one embodiment, tissue slides were placed in 20 mM sodium citratesolution (pH 8.5) around 100° C. and were cooled down in 60° C.incubation chamber for 30 mins.

Immunohistochemistry

Once the clinical tissues samples have been de-paraffinized andsubjected antigen retrieval, as necessary, the clinical tissue samplesare then immunostained by any method known to one skilled in the art. Insome embodiments, samples are first blocked with MAXBLOCK™ BlockingMedium (Active Motif) for 1 hour at 37° C., followed by incubation withprimary antibodies in MAXSTAIN™ Staining Medium (Active Motif) at aconcentration of 10 μg/mL for about 1 minute to about several days atabout 0° C. to about 40° C. depending on tissue thickness and antibody.In some embodiments, the clinical tissue samples are incubated with theprimary antibodies for 6-24 hours. In some embodiments, the clinicaltissue samples are incubated with the primary antibodies at about roomtemperature to about 37° C. In some embodiments, the clinical tissuesamples are incubated with the primary antibodies at about roomtemperature. In some embodiments, the clinical tissue samples areincubated with the primary antibodies at about 37° C. The clinicaltissues samples are then washed in MAXWASH™ Washing Medium (ActiveMotif) four times, for 5-30 minutes each, changing solutions in between.The clinical tissue samples are then incubated with appropriatesecondary antibodies at a concentration of approximately 10 μg/mLtogether with 300 nM DAPI in MAXSTAIN™ Staining Medium for about 1minute to about several days at about 0° C. to about 40° C. depending ontissue thickness and antibody. In some embodiments, the clinical tissuesamples are incubated with the primary antibodies for 6-24 hours. Insome embodiments, the clinical tissue samples are incubated with theprimary antibodies at about room temperature to about 37° C. In someembodiments, the clinical tissue samples are incubated with the primaryantibodies at about room temperature. In some embodiments, the clinicaltissue samples are incubated with the primary antibodies at about 37° C.The clinical tissue samples are then washed in MAXWASH™ Washing Mediumseveral times.

Chemical Treatment for Protein Preservation

Once the clinical tissues samples have been de-paraffinized andsubjected antigen retrieval, as necessary, and the clinical tissuesamples are immunostained, the samples can be treated for proteinperseveration as described by in WO 2017/027368, which is incorporatedherein by reference. In some embodiments, the clinical tissue sample istreated by incubation in PBS buffer containing 0.03-0.2 mg/ml Acryloyl XAcryloyl-X, SE (6-((acryloyl)amino) hexanoic acid, succinimidyl ester,here abbreviated AcX; (Thermo Fisher Scientific), for 2-12 hours.

In one embodiment, AcX was dissolved in anhydrous DMSO at aconcentration of 10 mg/mL, aliquoted and stored frozen in a desiccatedenvironment. Tissue slides were incubated with 0.03-0.1 mg/ml AcX (0.03mg/ml for samples fixed with non-aldehyde fixatives, 0.1 mg/ml forsamples fixed with aldehyde fixatives) diluted in PBS buffer for morethan 6 hours at RT.

In Situ Polymer Synthesis

Once the clinical tissues samples have been de-paraffinized andsubjected antigen retrieval, as necessary, and the clinical tissuesamples are immunostained and, optionally, treated for proteinperseveration, the clinical tissue samples are subjected to in situpolymer synthesis. Briefly, monomer solution including 1× PBS, 2 M NaCl,8.625% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, 0.10% (w/w)N,N′-methylenebisacrylamide (all from Sigma Aldrich), was prepared andaliquoted prior to in situ polymer synthesis. Tissue slides wereincubated with the monomer solution for about 1 hour at 4° C. to allowcomplete diffusion of monomer solution and prevent premature gelation.4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (Sigma Aldrich) asinhibitor, tetramethylethylenediamine as accelerator and ammoniumpersulfate as initiator, were added sequentially to the monomer solutionup to 0.2% (w/w) each. Finally, samples were incubated for 1.5-2 hour at37° C. with humidified atmosphere to complete gelation.

In some embodiments, a monomer solution including sodium acrylate,acrylamide, and bisacrylamide, salt and buffer is prepared prior to insitu polymer synthesis. The monomer solution may be cooled at 4° C. toprevent premature gelation. Ammonium persulfate as initiator,tetramethylethylenediamine as accelerator and4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl as inhibitor are added tothe monomer solution up to 0.2% (w/w) each. Tissue slides are incubatedwith the monomer solution at 4° C. (for variable time depending onthickness) to allow monomer solution to diffuse, and then incubate in37° C. with humidified atmosphere for 1-2 hour to enable gelation.

After the completion of polymerization, the regions of interest are cutusing razor blades or appropriate tool (For slide samples, the tissuesremain attached to the slides). In some embodiments, the regions ofinterest from samples can be cut out after the sample has been expanded.In some embodiments, the regions of interest from samples are cut outprior to expansion.

Sample Digestion and Expansion

Once the clinical tissues samples have been subjected to in situ polymersynthesis, the clinical tissue samples are incubated in 4-32 U/mlproteinase K (New England Biolabs) in a modified digestion buffercomprising 50 mM Tris (pH 8), 5-100 mM EDTA, 0.25% Triton X-100, and 0.4M guanidine HCl. In some embodiments, the clinical tissue samples areincubated at 50° C. for at least 8 hours until the completion ofdigestion.

In some embodiments, the clinical tissue samples are hard to digest, asregular buffers for proteinase K digestion do not work well withclinical tissue samples; thereby, making it difficult to ensure reliabledigestion of the sample. Thus, the invention also describes a “digestionduring expansion” method to overcome this problem. In some embodiments,the clinical tissue samples and the proteinase K are incubated in amodified digestion buffer comprising 50 mM Tris (pH 8), 25 mM EDTA,0.25% Triton X-100, and 0.4 M guanidine HCl. The salt concentration ismaintained at a low ionic strength which promotes moderate expansion ofthe tissue sample during the digestion and enables better penetration ofdigestive enzyme inside the tissue. To further aid digestion, highconcentration of disodium salt of ethylenediaminetetraacetic acid (EDTA)is used to chelate any residual divalent cation that maintains thestructural integrity of structural proteins in the tissues, such ascollagen and fibronectin. The incubation temperature is set to increaseenzyme activities.

In some embodiments, samples were incubated in 8 U/ml proteinase K (NewEngland Biolabs) in a modified digestion buffer containing 50 mM Tris(pH 8), 25 mM EDTA, 0.25% Triton X-100, 0.4 M guanidine HCl (or 0.4 MNaCl), and the tissues were incubated for 0.5-3 hours at 60° C. or untilthe completion of digestion. Digested samples were washed once with 1×PBS buffer and stained with 300 nM DAPI in PBS buffer for 1 hour, thenwashed at least twice with 1× PBS for at least 20 minutes each wash.Finally, gels were placed in doubly deionized water for 10 mins toexpand. This step was repeated 3-5 times in fresh water or 0.002% ˜0.01% sodium azide solution (to prevent bacterial growth), until thesize of the expanding sample remained unchanged.

In some embodiments, the completion of digestion of the clinical tissuesamples results in loose detachment of the tissues on the slides. Insome embodiments, the clinical tissue samples can be separated from theslides. In some embodiments, the clinical tissue samples can be stainedwith DAPI or other chemical nucleus stains. In one embodiment, theclinical tissue samples are washed once with 1× PBS buffer afterdigestion and stained with 300 nM DAPI in PBS buffer for 1 hour, thenwashed at least twice with 1× PBS for at least 20 minutes each wash. Ifthe samples are stained with DAPI or other chemical nucleus stainsbefore the in situ polymer synthesis, the stain would diffuse away inthe digestion buffer. To recover the nucleus staining, the samples arewashed once with 1× PBS buffer after digestion and stained with 300 nMDAPI in PBS buffer for 1 hour, then washed at least twice with 1× PBSfor at least 20 minutes each wash. Finally, the sample is expanded byiterative washing with Milli Q water.

DNA FISH

In some embodiments, for expanded samples being processed further forDNA FISH, the digested gel samples were placed in the hybridizationbuffer containing 1× PBS, 15% ethylene carbonate, 20% dextran sulfate,600 mM NaCl and 0.2 mg/ml single stranded salmon sperm DNA at 85° C. for30 mins, then mixed with 30 μL of hybridization buffer containingSureFISH probes (Agilent/Dako) pre-heated at 85° C. for 10 mins. Themixtures were then incubated at 45° C. overnight. The next day, thesamples were washed with stringency wash buffer containing 1×SSC (150 mMNaCl, 15 mM sodium citrate, pH 7.0) and 20% ethylene carbonate at 45° C.for 15 min, followed by a wash with 2×SSC at 45° C. for 3 times 10 minseach. Finally, the gel samples were washed with 0.02×SSC multiple times(5 mins each) until the expansion was completed.

Imaging

In some embodiments, imaging of the expanded clinical tissue samples canbe done with a conventional fluorescent, confocal microscope, or otherdesired scopes. Pre- and post-expansion images of clinical tissuessamples were acquired on an Andor Revolution Spinning Disk Confocal byplacing the pre- and post-expanded clinical tissue samples inglass-bottom six-well plates (In Vitro Scientific) and held in place bymounting with 1% agarose. Images were taken on a 40×1.10 NA (Nikon)water objective with 1× or 1.5× zoom and expansion of aparaffin-embedded tissue microarray is shown in FIG. 2 and reuse andexpansion of an H&E stained slide is shown in FIG. 4.

Fluorescent Microscopy After Expansion

Low-magnification images of specimens were imaged on a Nikon Ti-Eepifluorescence microscope with a SPECTRA X light engine (Lumencor), anda 5.5 Zyla sCMOS camera (Andor), controlled by NIS-Elements AR software,with a 4×0.13 NA air objective or 10×0.2 NA air objective (Nikon). Forsome images, the images were acquired on the same microscope with a40×1.15 NA water immersion objective (Nikon). The following filter cubes(Semrock, Rochester, N.Y.) were used: DAPI, DAPI-11LP-A-000; Alexa Fluor488, GFP-1828A-NTE-ZERO; Alexa Fluor 546, FITC/TXRED-2X-B-NTE; Atto 647Nor CF 633, Cy5-4040C-000.

Otherwise, all other presented fluorescent images were imaged using anAndor spinning disk (CSU-X1 Yokogawa) confocal system on a Nikon TI-Emicroscope body, with a 40×1.15 NA water immersion objective. DAPI wasexcited with a 405 nm laser, with 450/50 emission filter. Alexa Fluor488 was excited with a 488 nm laser, with 525/40 emission filter. AlexaFluor 546 was excited with a 561 nm laser with 607/36 emission filter.Atto 647N and CF633 were excited with a 640 nm laser with 685/40emission filter.

Brightfield Microscopy

Low magnification images were acquired on a Nikon Ti-E microscope with aDS-Ri2 sCMOS 16 mp Color Camera (Nikon) and white LED light, with a4×0.13 NA air objective or 10×0.2 NA air objective. High magnificationimages of H&E slides were acquired on the Pannoramic Scan II(3DHistech), with a 40×0.95 NA air objective (Zeiss).

Autofluorescence Analysis

Tissue-unrelated background was removed from all images by subtractionof mean pixel values from blank regions, prior to analysis. For eachfluorescent channel, 10 regions of interest containing brightestfluorescent signals and one area containing autofluorescence signal,judged by a pathologist's visual inspection, were selected, and used tocalculate signal-to-background ratios.

Measurement Error Quantification

The same fields of view in different z planes were first imaged pre- andpost-expansion. To match the z planes pre- and post-expansion,scale-invariant feature transform (SIFT) keypoints were generated forall the possible combination of pairs of pre- and post-expansion zplanes (or z projections). SIFT keypoints were generated using theVLFeat open source library and filtered by random sample consensus(RANSAC) with a geometric model limited to rotation, translation, andscaling. The pair of pre- and post-expansion images with the most SIFTkeypoints was used for image registration by rotation, translation anduniform scaling, as well as calculation of expansion factors and vectordeformation fields. By subtracting the resulting vectors at any twopoints, the entire population of possible measurements of point-to-pointlocalization error was sampled and the root-mean-square error for suchmeasurements was plotted as a function of measurement length.

Computational Nuclear Atypia Analysis

The following is a proposed framework for classification of expandedbreast tissue images into different categories: normal breast, benignbreast lesions (UDH and ADH) and non-invasive breast cancer. This imageclassification framework consists of four components: imagepreprocessing, nuclei segmentation, features extraction and imageclassification. The image pre-processing and nuclei segmentationpipelines are shown in FIG. 9A.

Image Pre-Processing

After tissue expansion and biomarker staining of nuclei with DAPI, imageacquisition was performed using confocal microscopes at 40Xmagnification. Due to acquisition of multiple non-overlapping tiles andstitching to produce a single image, these tiles demonstrated rollingball background noise. During image pre-processing, a rolling-ballbackground correction algorithm with ball size as average nuclei sizewas applied to remove uneven background noise. After the backgroundnoise removal, a nucleus to background contrast was enhanced by adaptivehistogram equalization. These enhanced images were smoothed by medianfilter of radius 10.

Nuclei Segmentation

The nuclei segmentation procedure consists of three steps. First, nucleiwere segmented using a Poisson distribution based minimum errorthresholding method. Standard and global thresholding methods are notefficient as minimum error threshold method, because of high variabilityin nuclei and background regions. In order to address this issue, thislocally adaptive thresholding algorithm selected the threshold bymodelling the mixture of two Poisson models using the image histogram.The threshold value was computed by minimizing the relative entropybetween the image histogram and the Poisson mixture model. The initialsegmentation of nuclei was then improved by a set of morphologicaloperations that include hole-filing and morphological closing to fillholes and combine small fragments of nuclei to form one single nuclei,and morphological opening to remove small non-nuclei regions (e.g. bloodvessels, parts of fragmented nuclei and artifacts). This segmentationmethod may under-segment clustered nuclei that are touching each other.Second, to separate the touching and overlapping nuclei, we usedscale-adaptive multi-scale Laplacian of Gaussian (MSLoG) filter toproduce local maxima and select seed points for nuclei. In the case ofselecting the local maxima points, the constant scale produces imprecisenuclear seed points, since the nuclear size varies considerably in earlybreast neoplasia lesions. In order to address this problem,scale-adaptive MSLoG filter was applied on a given number of scales andlocal maximum points in the scale-space response were selected as seedpoints. Last, these seed points were used as markers for themarker-controlled watershed algorithm to separate touching andoverlapping nuclei.

Feature Extraction

After nuclei segmentation, morphological, first-order statistical andsecond-order statistical features were extracted for each nucleus. Themorphological features include shape and geometrical features whichreflect the phenotypic information of nuclei. The computed morphologicalfeatures are area, convex area, perimeter, equivalent perimeter,eccentricity, orientation, solidity, extent, compactness, major axislength, minor axis length, elliptical minor and major radius. Thefirst-order statistical features determined the distribution ofgray-level values within the nuclei regions. The computed first-orderstatistical features are mean, median, mean absolute deviation, standarddeviation, interquartile range, skewness and kurtosis. The second-orderstatistical features determined the variation inside nuclei texture.

Two types of second order statistical features were computed using greylevel Haralick co-occurrence and run-length matrices. The co-occurrencematrix GLCM (i,j; d,θ) is square with dimension Ng where Ng is the totalnumber of grey levels in the image. The value at ith and jth column inthe matrix was produced by counting the total occasions a pixel withvalue i is adjacent to a pixel with value j at a distance d and angle θ.Then the whole matrix was divided by the total number of suchcomparisons that have been made. Alternatively, each element of GLCMmatrix is considered as the probability that a pixel with grey level iis to be found with pixel with grey level j at a distance d and angle θ.Adjacency was defined in four directions (vertical, horizontal, left andright diagonals) with one displacement vector, which produced four GLCMsmatrices. Texture information is rotationally invariant. So, the averagein all four directions was taken and produced one GLCM matrix. Later, 14features proposed by Haralick were computed from the GLCM in order toidentify texture more compactly. These 14 features are Autocorrelation,Correlation, Contrast, Cluster Shade, Cluster Prominence, Energy,Entropy, Homogeneity, Inverse Difference Normalized, Inverse DifferenceMoment Normalized, Dissimilarity, Maximum Probability, InformationMeasure Correlation 1 and Information Measure Correlation 2.

The set of consecutive pixels, with the same grey level, collinear in agiven direction, constitutes a grey level run length matrix GLRLM (i,j;θ). The dimension of GLRLM is Ng×R, where Ng is the number of greylevels and R is the maximum run length. Similar to the GLCM, GLRLMs werecomputed for four directions and later average them. The 11 run-lengthfeatures, derived from GLRLM, are short run emphasis (SRE), long runemphasis (LRE), grey-level non-uniformity (GLN), run lengthnon-uniformity (RLN), ratio-percentage (RP), low grey level runsemphasis (LGLRE), high grey level runs emphasis (HGLRE), short run lowgrey level emphasis (SRLGLE), short run high grey level emphasis(SRHGLE), long run low grey level emphasis (LRLGLE) and long run highgrey level emphasis (LRHGLE). In total, 45 features were computed foreach nucleus. Last, these features were summarized at the image level bycomputing the first-order statistics including mean, median, meanabsolute deviation, standard deviation, interquartile range, skewnessand kurtosis of each feature per image, producing 315 summary featuresper image.

Image Classification

During the last part of the framework, logistic regression was performedwith Lasso regularization to build multivariate image feature-basedmodels to classify normal, benign and pre-invasive malignant tissueimages. The analyses were implemented in R, using the glmnet package.Lasso regularization was used to create simpler models, less prone tooverfitting, than those that would be obtained from standard logisticregression. The Lasso procedure consists of performing logisticregression with an L1 regularization penalty, which has the effect ofshrinking the regression weights of the least predictive features to 0.The amount of the penalty (and the number of non-zero features in themodel) is determined by the regularization parameter λ. This method hasbeen shown to perform well in the setting of colinearity and has beenwidely used to build predictive models from high-dimensional data intranslational cancer research. Features were standardized separately inthe training and validation data-sets prior to model construction, usingthe selected setting in glmnet. Model performance with 6 fold crossvalidation (6F-CV) was evaluated. For validation, the value of λ thatachieved the maximum area under curve (AUC) in cross-validation wasselected on the training fold data-set and applied this fixed model tothe validation fold data-set. Model performance was assessed bycomputing the area under the receiver operator curve (AUC) of truepositives vs. false positives, where a perfect classifier would achievean AUC of 1, while a random classifier would achieve an AUC of 0.5. Theevaluation of the framework was also performed using two other machinelearning classifiers, which are commonly used in biomedical research. Arandom forest classifier fits a number of decision trees on varioussub-samples of the dataset and use averaging to improve the predictiveaccuracy and control over-fitting. Number of trees (numTrees), maximumdepth of the tree (maxDepth) and number of features (numFeatures) to beused in random selection are three parameters that affect the randomforest performance. In the experiments numTrees=100, maxDepth=30 andnumFeatures=20 were used. The last classifier is Naïve Bayes, which is aprobabilistic classifier based on applying Bayes' theorem with strongindependence assumptions between the features. As the predicted value isclass label (e.g., we are pursuing a classification problem), theindependence assumption is less restrictive for classification ascompared to regression.

Image Classification Results

Image classification framework was applied on both pre-expanded andexpanded images. Both data sets consist of 105 images containing 36normal breast tissue images, 31 non-invasive lesion breast tissue images(15 UDH and 16 ADH) and 38 pre-invasive breast tissue images (DCIS). Sothese 105 images belong to 4 different classes (Normal, UDH, ADH andDCIS). The total number of cases was 131; 105 cases were analyzed and 26were excluded because they were judged to be borderline diagnosticcases. In order to discriminate normal breast tissue with eachnon-invasive and pre-invasive breast tissue types, binary classificationwas performed for all classes and results are shown in FIG. 9C. In orderto discriminate normal breast tissue with UDH, ADH and DCIS tissue,GLMNET classifier reported AUC 0.95, 0.96 and 0.94 on expanded data ascompared to AUC 0.86, 0.82 and 0.75 with pre-expanded data,respectively. For differentiating non-atypical breast tissue (UDH) fromatypical breast tissues (ADH and DCIS), GLMNET classifier reported AUC0.93 and 0.89 on expanded data as compared to AUC 0.71 and 0.82 withpre-expanded data, respectively. For discriminating atypical benignbreast tissue (ADH) with pre-invasive breast cancer tissue (DCIS),GLMNET classifier reported AUC 0.95 with expanded data as compared toAUC 0.84 with pre-expanded data.

Clinical Samples and Pathology-Optimized Expansion Microscopy

Three starting states for clinical and pathological tissue samples wereconsidered when devising a series of steps so that different clinicalsamples would all arrive in a condition optimized for ExM processing(FIG. 1A): formalin fixed paraffin-embedded (FFPE), H&E stained tissuesections, and fresh frozen tissue, all assuming the tissue to bethin-sliced and on a glass slide. FFPE samples were tested first sinceit was hypothesized that all of the steps required for the other threecategories would either be subsets or permutations of the steps requiredfor FFPE tissue processing. It was evaluated whether xylene treatment toremove paraffin, followed by rehydration and a fairly standard antigenretrieval step (e.g., placing samples in 20 mM sodium citrate at pH 8and 100° C., and then immediately transferring to a 60° C. chamber for30 mins), could enable samples to be processed. It was found thatheavily formalin-fixed human tissues did not expand evenly under theprotocol, even after paraffin removal, unless digestion was performed.The effects of EDTA at 1 mM vs. 25 mM concentration in the digestion ofhuman samples including skin, liver, breast and lung, includingacetone-fixed as well as FFPE samples (FIG. 3; Table 2) was examined.

TABLE 2 The effects of EDTA concentration on proteinase K digestion ofhuman tissue/hydrogel hybrid samples. Digestion* with Digestion with 1mM EDTA 25 mM EDTA Fresh Frozen FFPE Fresh Frozen FFPE Skin

Liver

Lung

Breast

*Digestion condition: 8 units/mL proteinase K solution containing 25 mMTris (pH 8), 0.25% Triton X-100, 0.4M NaCl, in 60° C. for 3 hours.

 Complete digestion.

 Incomplete digestion.

The effect of digestion time under both conditions for human skin andliver FFPE samples, which contain distinct extracellular matrixcomponents and exhibit strong autofluorescence in the blue and greenfluorescence emission channel due to formalin-fixed extracellular matrix(ECM) proteins, was investigated.

TABLE 3 The effects of EDTA concentration on proteinase K digestion ofhuman tissue/hydrogel hybrid samples as a function of digestion time.Human EDTA Digestion time tissue type concentration 0.5 h 1 h 1.5 h 2 hSkin 1 mM

25 mM

Liver 1 mM

25 mM

It was found that both human skin and liver samples were completelydigested and fully expanded within 0.5 hour with 25 mM EDTA-assistedproteinase K digestion. On the other hand, the ECM was not fullydigested in either type of tissue with 1 mM EDTA as indicated byresidual autofluorescence (FIG. 3Aiii, Avi). Incomplete digestion cancause distortions at both micro- and macro-scales. As for other types oftissues, both methods suffice.

This FFPE pipeline, with xylene treatment and increased EDTA, couldprepare samples for the methods described herein, was validated byassessing the entire pipeline on normal human breast tissues preparedwith FFPE preservation. Pre-expansion imaging with either a widefield(FIG. 1B) or SR-SIM (FIG. 1F) microscope, followed by post-expansionimaging on widefield (FIG. 1C) or confocal (FIG. 1G) microscopesrespectively, yielded low distortion levels of a few percent (FIGS. 1D,1E, 1H, and 1I). Thus, this expansion pathology (ExPath), protocol wasable to expand paraffin embedded and highly aldehyde-fixed samples.

Having established a basic ExPath pipeline, it was desirable to prepareH&E-stained samples as well. For mounted samples, the cover slip andmounting medium (a hard substance, made out of polystyrene) had to beremoved; since xylene treatment was acceptable as a pre-treatment forexpansion microscopy, a xylene pre-treatment step was added to dissolveaway the mounting medium and result in coverslip removal.

H&E stained tissues exhibit high background fluorescence, suggestingthat removal of eosin and hematoxylin would be important for laterfluorescent antibody staining. With the ExPath protocol, eosin andhematoxylin were both naturally removed over the time-course ofprocessing. Thus, it was demonstrated that mounted H&E samples could beprepared by visualizing nuclear DNA (stained with DAPI after digestion),as well as applying antibody stains against the mitochondrial proteinHsp60 and vimentin, using an H&E slide of human breast tissue withatypical ductal hyperplasia (ADH) (FIGS. 1J, 1K).

Finally, for fresh frozen sections preserved with acetone fixation; itwas found that lowering the concentration of AcX from 0.1 mg/mL to 0.03mg/mL enabled better processing (FIGS. 3K, 3L), perhaps because of thegreater number of free amines available in tissues that had not beenprocessed with aldehyde.

Having established the basic ExPath protocol, the protocol was extendedto experimental contexts. For example, DNA fluorescent in situhybridization (FISH) is commonly used to assess HER2 gene amplificationin breast cancer. DAPI was able to stain DNA post-expansion, consistentwith gel retention of DNA in expanded samples; therefore, it wasexamined whether post-expansion DNA FISH was possible. The large size oftraditional double-stranded bacterial artificial chromosome (BAC)-basedFISH probes (e.g. the length of BAC-based FISH probes targeting HER2 isapproximately 220 kb) precludes staining of expanded samples, socommercially available SureFISH probes, which are libraries ofsingle-stranded oligonucleotides with an average size of ˜150 bases,targeting HER2 and (as a control) the centrosome of chromosome 17 werechosen. It was observed that SureFISH probes diffused into breast ExPathsamples and hybridized with chromosomal DNA, for commercially availablespecimens of breast cancers with no amplification of HER2 (FIG. 1L) andfor cancer with HER2 amplification (FIG. 1M), with much more DNAhybridization apparent in the HER2-amplified case. Since DNA FISH isperformed as the final step of the process, it does not interfere withimmunostaining which occurs earlier in the protocol. Breast samples wereco-stained with an antibody against HER2 protein and confirmed thecorrelation of HER2 protein expression with HER2 gene amplification(FIGS. 1L, 1M).

ExPath, because it spaces apart molecules and also results inelimination of unwanted molecules, presents several advantages overconventional immunostaining. For example, tissue autofluorescenceremains challenging for clinical applications of immunofluorescence andfluorescence in situ hybridization in pathology analysis despite ofexisting autofluorescence reduction methods. Specimens processed withExPath are >99% water, and thus transparent and refractive index-matchedto water. Thus, the molecular clearing of ExPath, which eliminatesunanchored biomolecules (including potentially both proteins as well assmall molecules) that contribute to autofluorescence, has a verypractical outcome: namely, the reduction of autofluorescence, by anorder of magnitude in some spectral channels, as compared to the signal.

ExPath was applied to commercially available tissue microarrayscontaining dozens of samples from various organs, examiningcancer-containing vs. normal tissues from 8 different organs—breast,prostate, lung, colon, pancreas, kidney, liver and ovary, in all casesobtaining expansions of ˜4-5×, with average expansion factor 4.7(standard deviation (SD) 0.2). The expansion variation is smaller than10%, indicating consistent performance of expansion across differenttypes of human tissues. ExPath revealed sub-diffraction limitedstructures of intermediate filaments keratin and vimentin, which arecritical in the epithelial-mesenchymal transition, cancer progression,and initiation of metastasis (FIG. 2). Given that vimentin is a standardmarker of stromal tissue and keratin a standard marker of epithelialtissue, ExPath will provide a simple and convenient way to observesub-diffraction morphologies of not only nucleic acids, but also proteinbiomarkers, in clinical biopsy samples from a wide range of humanorgans.

ExPath Enables Visualization of Human Podocyte Tertiary Foot Processes

Given that nanoscale imaging has not become widespread in pathology, theExPath method described herein can be used in the exploration of normalvs. abnormal samples, followed by traditional or automated inspection ofkey features, both for pinpointing novel pathological mechanisms, aswell as for disease classification and refined diagnosis. For example,kidney diseases such as minimal change disease (MCD) and focal segmentalglomerulosclerosis (FSGS) (and other lesions associated with nephroticsyndrome) are typically diagnosed or confirmed via electron microscopy.In MCD, kidney tertiary podocyte foot processes, which typically coverthe surface of glomerular capillary loops like interdigitating fingers,instead lose their characteristic morphology and appear continuous underelectron microscopy—a phenomenon called foot process effacement (FPE).The width of individual foot processes is around 200 nm, beyond thelimits of resolution using conventional optical microscopy. It wasdetermined whether ExPath could help with visualization of podocyte footprocesses. First a protein target that is specific and abundant intertiary podocyte foot processes was identified, examining theimmunofluorescence of a selection of reported podocyte foot processmarkers. Among the potential protein targets to show specific andintense podocyte foot process staining, in the ExPath context foracetone-fixed frozen kidney samples that were heat treated prior toimmunostaining (FIGS. 6 and 7) was actinin-4. Also identified wasanti-synaptopodin²³ antibody suitable for ExPath imaging (FIG. 7). Thequality of immunostaining of anti-actinin-4 decreased slightly on kidneyFFPE samples treated with either citrate or Tris-EDTA antigen retrievalmethods (FIG. 8), compared to that of acetone-fixed frozen kidneysamples. The decrease in immunostaining quality might be due to degradedantigenicity caused by formalin-induced crosslinking. Co-staining humankidney samples with anti-actinin-4, as well as antibodies againstvimentin (a glomerular marker), and collagen IV (a marker of thebasement membrane of capillaries), allowed for observation of themicroanatomy of glomeruli (FIG. 5A vs. B), with ExPath revealing theultrafine structures of tertiary podocyte foot processes (FIG. 5B) vs.conventional confocal imaging (FIG. 5A), in normal human kidney samples(FIG. 5C). Accordingly, fresh-frozen kidney sections from patients withnormal kidneys, as well as patients with MCD or FSGS, were stained andexpanded. ExPath images were acquired and compared to the electronmicroscopic images of the corresponding cases. Similar to results innon-clinical human kidney samples, the ultrafine structure of tertiaryfoot process in the kidneys from normal cases was observed (FIG. 5E),but foot process effacement was observed in MCD cases (FIG. 5G),consistent with the foot process morphologies seen in corresponding EMimages from the same samples (FIGS. 5D and 5F). Thus, the nanoscaledifferences between human clinical samples of nephrotic disease could bevisualized with conventional diffraction-limited optical microscopesafter sample expansion.

To examine in a blinded study whether ExPath could enable accurateidentification of foot process effacement from both MCD and FSGS cases,seven observers, including five pathologists and two non-pathologists,first studied a sample set of immunofluorescent images of kidneyglomeruli in both pre-expansion and post-expansion states, and thenexamined 10 different pre-expansion and 10 post-expansionimmunofluorescence images of kidney glomeruli from 3 specimens fromnormal subjects, 2 from MCD patients and 1 from an FSGS patient. Forunexpanded samples, the classification accuracy was only 65.7% (standarddeviation (SD) 17%), but increased significantly to 90% (SD 8%) ifpost-expansion images were used instead (p=0.0088, two tailed t-test).To assess the inter-observer agreement, Fleiss's kappa values werecalculated for observers' categorical ratings on both sets of data.Strong consistency on observers' ratings of post-expansion data werefound, with kappa value 0.68±0.14 at the 95% confidence level, whileinter-observer agreement on pre-expansion data (0.35±0.13, 95%confidence level) was poor—indeed, the kappa value was borderline, giventhe clinically acceptable threshold of 0.40. ExPath enabled accurate andconsistent evaluation among observers on whether the image was from asample in a normal or abnormal state, from a single post-expansion image(in clinical practice, of course, kidney pathologists normally examinemultiple selected EM images for accurate diagnosis). The results suggestthat large-scale blinded studies using ExPath may be highly relevant forstreamlining the diagnosis or confirmation of nephrotic kidney disease,and potentially other diseases that involve known nanoscale pathology,as well as helping detect diseases earlier when the changes are toosmall to be resolved with ordinary microscopes.

ExPath Significantly Improves Computational Diagnosis in Early BreastLesions

One of the most challenging problem areas in breast pathology is theclassification of early breast lesions. For example, one study has shownthat the concordance rate between expert pathologists and pathologistspracticing in the community can be quite variable in the classificationof non-invasive lesions of the breast, with an agreement of only about50% for atypical breast lesions¹¹. The pathological classification ofthese lesions provides critically important diagnostic information toprevent over- and under-treatment, and to guide clinical management.

It was hypothesized that the problems with the current classificationschemes are due to two issues: first, the diagnostic criteria arelargely qualitative and subjective; second, the information contained inthe images is limited by the optical diffraction limit of conventionaloptical microscopes. To begin to address the first issue, computationalpathology models were developed that can discriminate benign frommalignant intraductal proliferative breast lesions. However, theefficacy of these models is limited by the information extractable fromdiffraction-limited images. Therefore, ExPath was used to examine thepathological classification of early breast lesions since the extrainformation obtained from expanded samples might lead to a higherquality of extracted features, in turn resulting in improvements in thereproducible classification of pre-invasive breast lesions.

An automated segmentation framework was applied on pre-expandedH&E-stained images, as well as an image classification framework updatedwith nucleus detection and segmentation algorithms optimized forpost-expansion DAPI-stained images (FIG. 9A). The image classificationframework for post-expansion DAPI-stained images includes foregrounddetection, nucleus seed detection, and nuclear segmentation (FIG. 9A).Following application of the framework, three kinds of features wereextracted from each segmented nucleus from both the pre-expanded andpost-expanded images: nuclear morphology features, nuclear intensityfeatures, and nuclear texture features.

Each of the two datasets (pre- and post-expansion) consists of 105images: 36 normal breast tissue images, 31 proliferative lesion (benign)images (15 usual ductal hyperplasia (UDH), 16 atypical ductalhyperplasia (ADH)) and 38 ductal carcinoma in situ (DCIS). All sampleswere expanded by ˜4-5×, with average expansion factor of 4.8 (SD: 0.3).The impact of ExPath on the performance of nuclear detection andsegmentation algorithms for a subset of 31 images from bothpre-expansion and expanded datasets was assessed (6 normal, 9 UDH, 9 ADHand 7 DCIS; FIG. 9B). Computational detection of nuclei wassignificantly more accurate in expanded samples (FIG. 9B), with an 11%increase in true positive rate, 22% increase in positive predictivevalue, and 16% increase in f-score, over non-expanded samples, andsegmentation was significantly improved as well, with a 14% increase inf-score, 77% increase in kappa and 66% decrease in global consistencyerror (GCE). This improved accuracy of nuclear detection andsegmentation will help support improved computational pathologyanalyses. To this end, the classification performance was improved whenthe models were run on post-expansion data, as compared withpre-expansion data (FIG. 9C). In particular, when examining the areaunder the receiver operator curve (AUC) of true positives vs. falsepositives—where a perfect classifier would achieve an AUC of 1, while arandom classifier would achieve an AUC of 0.5—the pipeline was able todiscriminate lesions such as UDH from atypical lesions such as ADH withan AUC of 0.93 on expanded samples, compared with only 0.71 on thepre-expanded tissue.

These findings suggest that the improved nuclear segmentation achievedon post-expansion images results in more informative features that inturn result in higher-performing classification models. Thus, ExPath canfacilitate computational pathology differentiation of proliferativebreast lesions, providing diagnostic information which could potentiallyprevent over- and under-diagnosis and guide clinical management.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed:
 1. A method for examining podocyte foot processescomprising the steps of (a) staining a kidney tissue sample with footprocess-specific antibodies; (c) subjecting the sample to microscopicanalysis.
 2. The method according to claim 1, wherein the kidney tissuesample is an expandable sample.
 3. The method according to claim 2,wherein the expandable sample is prepared by (a) contacting the samplewith a bi-functional linker; (b) permeating the sample with precursorsof a swellable material (c) polymerizing the precursors to form aswellable polymer within the sample, wherein the bi-functional linkerattaches (or crosslinks) to the swellable material; (d) incubating thesample with a non-specific protease in a buffer comprising a metal ionchelator, a nonionic surfactant, and a monovalent salt.
 4. The methodaccording to claim 3, wherein the bi-functional linker attaches to theswellable material during polymerization.
 5. The method according toclaim 3, wherein the bi-functional linker attaches to the swellablematerial after polymerization.
 6. The method according to claim 3,wherein the bi-functional linker comprises a protein-reactive chemicalmoiety and a gel-reactive chemical moiety.
 7. The method according toclaim 6, wherein the protein-reactive chemical moiety is a succinimidylester of 6-((acryloyl)amino) hexanoic acid (AcX)
 8. The method accordingto claim 3, further comprising expanding the sample by contacting theswellable polymer with a solvent or liquid to cause the swellablepolymer to swell.
 9. The method according to claim 1, wherein the sampleis a previously preserved clinical sample.
 10. The method according toclaim 9, wherein the sample is a formalin fixed paraffin embedded (FFPE)or a hematoxylin and eosin (H&E) stained tissue sample, or a freshfrozen sample.
 11. The method according to claim 10, wherein prior toexamining the sample is subjected to (i) de-coverslipping the sample ifit is mounted; (ii) treating the sample to mounting medium removal;(iii) treating the sample to re-hydration if step (ii) is performed; and(iv) subjecting the sample to antigen-retrieval.
 12. The methodaccording to claim 3, wherein the sample is incubated with about 1 toabout 100 U/ml of a non-specific protease in a buffer having a pHbetween about 4 and about 12, the buffer comprising about 5 mM to about100 mM of a metal ion chelator; about 0.1% to about 1.0% of a nonionicsurfactant; and about 0.05 M to about 1.0 M monovalent salt.
 13. Amethod for diagnosing kidney disease comprising the steps of (a)staining a kidney tissue sample with foot process-specific antibodies;and (b) subjecting the sample to microscopic analysis.
 14. The methodaccording to claim 13, wherein the kidney tissue sample is an expandablesample.
 15. The method according to claim 14, wherein the expandablesample is prepared by (a) contacting the sample with a bi-functionallinker; (b) permeating the sample with precursors of a swellablematerial (c) polymerizing the precursors to form a swellable polymerwithin the sample, wherein the bi-functional linker attaches (orcrosslinks) to the swellable material; (d) incubating the sample with anon-specific protease in a buffer comprising a metal ion chelator, anonionic surfactant, and a monovalent salt.
 16. The method according toclaim 15, wherein the bi-functional linker attaches to the swellablematerial during polymerization.
 17. The method according to claim 15,wherein the bi-functional linker attaches to the swellable materialafter polymerization.
 18. The method according to claim 15, wherein thebi-functional linker comprises a protein-reactive chemical moiety and agel-reactive chemical moiety.
 19. The method according to claim 18,wherein the protein-reactive chemical moiety is a succinimidyl ester of6-((acryloyl)amino) hexanoic acid (AcX).
 20. The method according toclaim 15, further comprising expanding the sample by contacting theswellable polymer with a solvent or liquid to cause the swellablepolymer to swell.
 21. The method according to claim 13, wherein thesample is a previously preserved clinical sample.
 22. The methodaccording to claim 21, wherein the sample is a formalin fixed paraffinembedded (FFPE) or a hematoxylin and eosin (H&E) stained tissue sample,or a fresh frozen sample.
 23. The method according to claim 22, whereinprior to examining the sample is subjected to (i) de-coverslipping thesample if it is mounted; (ii) treating the sample to mounting mediumremoval; (iii) treating the sample to re-hydration if step (ii) isperformed; and (iv) subjecting the sample to antigen-retrieval.
 24. Themethod according to claim 15, wherein the sample is incubated with about1 to about 100 U/ml of a non-specific protease in a buffer having a pHbetween about 4 and about 12, the buffer comprising about 5 mM to about100 mM of a metal ion chelator; about 0.1% to about 1.0% of a nonionicsurfactant; and about 0.05 M to about 1.0 M monovalent salt.
 25. Amethod for analyzing foot process effacement comprising the steps of (a)staining a kidney tissue sample with foot process-specific antibodies;and (b) subjecting the sample to microscopic analysis.
 26. The methodaccording to claim 25, wherein the kidney tissue sample is an expandablesample.
 27. The method according to claim 26, wherein the expandablesample is prepared by (a) contacting the sample with a bi-functionallinker; (b) permeating the sample with precursors of a swellablematerial (c) polymerizing the precursors to form a swellable polymerwithin the sample, wherein the bi-functional linker attaches (orcrosslinks) to the swellable material; (d) incubating the sample with anon-specific protease in a buffer comprising a metal ion chelator, anonionic surfactant, and a monovalent salt.
 28. The method according toclaim 27, wherein the bi-functional linker attaches to the swellablematerial during polymerization.
 29. The method according to claim 27,wherein the bi-functional linker attaches to the swellable materialafter polymerization.
 30. The method according to claim 27, wherein thebi-functional linker comprises a protein-reactive chemical moiety and agel-reactive chemical moiety.
 31. The method according to claim 30,wherein the protein-reactive chemical moiety is a succinimidyl ester of6-((acryloyl)amino) hexanoic acid (AcX).
 32. The method according toclaim 27, further comprising expanding the sample by contacting theswellable polymer with a solvent or liquid to cause the swellablepolymer to swell.
 33. The method according to claim 25, wherein thesample is a previously preserved clinical sample.
 34. The methodaccording to claim 33, wherein the sample is a formalin fixed paraffinembedded (FFPE) or a hematoxylin and eosin (H&E) stained tissue sample,or a fresh frozen sample.
 35. The method according to claim 34, whereinprior to examining the sample is subjected to (i) de-coverslipping thesample if it is mounted; (ii) treating the sample to mounting mediumremoval; (iii) treating the sample to re-hydration if step (ii) isperformed; and (iv) subjecting the sample to antigen-retrieval.
 36. Themethod according to claim 27, wherein the sample is incubated with about1 to about 100 U/ml of a non-specific protease in a buffer having a pHbetween about 4 and about 12, the buffer comprising about 5 mM to about100 mM of a metal ion chelator; about 0.1% to about 1.0% of a nonionicsurfactant; and about 0.05 M to about 1.0 M monovalent salt.